Preservative System For Acidic Beverages Based On Sequestrants

ABSTRACT

The present invention provides beverage preservative systems for use in high acid beverage products, and beverage products comprising the beverage preservative systems. The beverage preservative system prevents spoilage by microorganisms in a beverage within a sealed container for a period of at least 16 weeks. The present invention reduces or eliminates the use of conventional preservatives that pose health and/or environmental concerns. The components that make up the beverage preservative system of invention work together in a synergistic manner to reduce the amount of preservative required and so improve the inventive beverage&#39;s sensory impact over beverages having conventional preservatives.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 14/086,043, filed Nov.21, 2013, now U.S. Pat. No. 9,560,871, which is a divisional of U.S.Ser. No. 12/640,142 filed Dec. 17, 2009, now U.S. Pat. No. 8,628,812,which claims benefit from provisional application Ser. No. 61/141,481,now expired, all three cases being entitled Preservative System ForAcidic Beverages Based On Sequestrants. The above mentioned applicationsare incorporated herein by reference in their entireties.

TECHNICAL FIELD

This invention relates to beverage preservative systems and beverageproducts comprising the preservative system. In particular, thisinvention relates to beverage preservative systems having formulationssuitable to meet consumer demand for healthy and environmentallyfriendly ingredients.

BACKGROUND

Many food and beverage products include chemical preservatives to extendthe shelf-life of the product by inhibiting the growth of spoilagemicroorganisms (e.g., mold, yeast, bacteria) in the product for anextended period of time. However, some preservatives currently in usehave been found to have detrimental health and/or environmental effects,or are not sufficiently stable. Therefore, there is market demand forfood and beverage products which do not include these detrimentalpreservatives, and yet still possess extended shelf-life. There is alsoconsumer demand for natural ingredients in food and beverage products.

For example, benzoic acid and its salts are commonly used in beverageproducts as preservatives. However, benzoic acid and its salts can reactwith ascorbic acid (Vitamin C), to form benzene, which is a carcinogen.Heat and light increase the rate of this reaction, so production andstorage of beverage products under hot or bright conditions speeds upformation of benzene. Intake of benzene in drinking water is a publichealth concern, and the World Health Organization (WHO) and severalgoverning bodies including the United States and the European Union haveset upper limits for benzene content in drinking water of 10 ppb, 5 ppb,and 1 ppb, respectively.

Ethylenediamine tetraacetic acid (EDTA) and its salts is another commonbeverage product preservative. EDTA is a metal ion chelator thatsequesters metal ions and prevents their participation in catalyticoxidation reactions. EDTA at elevated concentrations is toxic tobacteria due to sequestration of necessary metals from the outermembrane of bacteria. However, EDTA is not bio-degradable, nor is itremoved during conventional wastewater treatment. Recalcitrant chelatingagents such as EDTA are an environmental concern predominantly becauseof their persistence and strong metal chelating properties. Widespreaduse of EDTA and its slow removal under many environmental conditions hasled to its status as the most abundant anthropogenic compound in manyEuropean surface waters. River concentrations in Europe are reported inthe range of 10-100 μg/L, and lake concentrations are in the range of1-10 μg/L. EDTA concentrations in U.S. groundwater receiving wastewatereffluent discharge have been reported in the range of 1-72 μg/L, andEDTA was found to be an effected tracer for effluent, with higherconcentrations of EDTA corresponding to a greater percentage ofreclaimed water in drinking water production wells.

The presence of chelating agents in high concentrations in wastewaterand surface water has the potential to remobilize heavy metals fromriver sediments and treated sludge, although low and environmentallyrelevant concentrations seem to have only a very minor influence onmetal solubility. Elevated concentrations of chelating agents enhancethe transport of metals (e.g., Zn, Cd, Ni, Cr, Cu, Pb, and Fe) in soilsand enhance the undesired transport of radioactive metals away fromdisposal sites. Low concentrations of chelating agents may eitherstimulate or decrease plankton or algae growth, while highconcentrations always inhibit activity. Chelating agents are non-toxicto many forms of life upon acute exposure; the effects of long-termlow-level exposure are unknown. EDTA ingestion at high concentrations bymammals changes excretion of metals and can affect cell membranepermeability.

Polyphosphates are another common beverage product preservative.However, polyphosphates are not stabile in aqueous solution and degraderapidly at ambient temperature. Degradation of polyphosphates results inunsatisfactory sensory issues in the beverage product, such as change inacidity. Also, the shelf-life of the beverage product is compromisedbecause of the reduced anti-microbial action from the reducedconcentration of polyphosphate.

It is therefore an object of the present invention to provide newpreservative systems for use in beverages as full or partialreplacements for at least one currently used preservative that hasdetrimental health and/or environmental effects, or lack of sufficientstability. It is further an object of the invention to provide newbeverage preservative systems with improved sensory impact. It isfurther an object of the invention to provide preservative systemswithout benzoic acid and/or reduced concentrations of sorbic acid. Somecountries have regulatory restrictions on the use of sorbic acid in foodand beverage products wherein the permitted concentration is less thanthe amount required to inhibit the growth of spoilage microorganisms byitself.

It is further an object of the invention to identify a sequestrant whichis bio-degradable and can be substituted for EDTA. It is further anobject of the invention to identify a sequestrant which is stable toheat in aqueous solution and can be substituted for polyphosphates. Theinvention is novel and unique in that only a very limited number ofknown compounds are able to complex metal ions so as to make the metalunavailable for use by spoilage microorganisms, but do not causemeasurable concern to health and nutrition experts. The odds that anysuch compound will also fall within the constraints on sensory impact ina beverage is on the order of 1:1000 to 1:10,000.

SUMMARY

According to a first aspect of the present invention, beveragepreservative systems are provided which comprise a sequestrant selectedfrom the group consisting of a biodegradable sequestrant,ethylenediamine tetraacetic acid (EDTA), a reverse sequestrant, aphosphonate, a polyphosphate, and mixtures of any of them; a weak acidselected from the group consisting of cinnamic acid, sorbic acid, theiralkali metal salts (e.g., Na⁺, K⁺), and mixtures of any of them; and apH of 5.8 or less; wherein the beverage preservative system preventsspoilage by microorganisms in a beverage within a sealed container for aperiod of at least 16 weeks.

In certain exemplary embodiments, the biodegradable sequestrant isselected from the group consisting of ethylenediamine-N,N′-disuccinicacid (EDDS), ethylenediamine-N,N′-dimalonic acid (EDDM),ethylenediamine-N,N′-diglutaric acid (EDDG), and mixtures of any ofthem. In this document, EDDS it is understood to be stereo isomer S,SEDDS as opposed to R,S or R,R

In certain exemplary embodiments the phosphonate is selected from thegroup consisting of phosphonic acids, bis-phosphonic acids,N-bis-phosphonic acids, their alkali metal salts (e.g., Na⁺, K⁺), andmixtures of any of them. In certain exemplary embodiments, thepolyphosphate is selected from the group consisting of sodiumhexametaphosphate (SHMP), sodium acid metaphosphate (SAMP), and mixturesthereof.

According to another aspect of the present invention, beverages areprovided which comprise a beverage component, the beverage preservativesystem as disclosed herein, and a pH of 5.8 or less, wherein thebeverage when placed within a sealed container is substantially notspoiled by microorganisms for a period of at least 16 weeks.

These and other aspects, features, and advantages of the invention or ofcertain embodiments of the invention will be apparent to those skilledin the art from the following disclosure and description of exemplaryembodiments.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a sequence of events for binding and releasing a tracemetal cation.

FIG. 2A depicts a yeast strain culture collection.

FIG. 2B depicts the pattern of spotting to gel-surface of plates.

FIG. 2C depicts a control plate and plates with organic acidpreservative

FIG. 2D depicts plates with cinnamic acid.

FIG. 3A depicts test results utilizing potassium sorbate.

FIG. 3B is a graph to facilitate interpretation of FIG. 3 a.

FIGS. 4A-4C depict data for the combination of potassium sorbate withEDTA (30 ppm), EDDS (30 ppm), and EDDS (15 ppm), respectively.

FIGS. 5A-5B data demonstrate the combination SHMP and potassiumcinnamate with EDTA and EDDS respectively.

FIGS. 6A-6D demonstrate the combination of EDDS & SHMP, EDTA & SHMP,EDDS & Eridonate, and EDTA and Eridonate, respectively.

FIGS. 7A, 7B, and 7C depict data collected from a hot-fill process.

DETAILED DESCRIPTION

The present invention is directed to beverage preservative systems andbeverage products comprising the preservative system. Among thecomponents of the beverage preservative system or beverage product ofinvention, none are able to individually inhibit the growth of spoilagemicroorganisms when present at concentrations employed in the presentinvention. Only when the components are assembled together in thepresent invention do they yield a cascade of bio-physical interactionsthat serve to disrupt the metabolism of spoilage microorganisms so as toprevent their outgrowth. The components of the invention do not justprovide an additive preservative effect, but work together in asynergistic manner to inhibit growth of spoilage microorganisms in abeverage within a sealed container for a period of at least 16 weeks. Byvirtue of the synergy between various components of the beveragepreservative system of invention, a lower concentration of eachcomponent is needed than would be the case if using conventionalpreservatives. Thus, flavor impact of the preservative system inbeverages can be reduced or minimized, and the beverage product ofinvention possesses surprisingly superior sensory impact, includingsuperior flavor, aroma, and quality, compared to beverages usingconventional preservatives.

Specifically, in certain exemplary embodiments, the biodegradablesequestrant or EDTA or the reverse sequestrant binds and sequesters atleast copper, nickel, chromium, and aluminum cations. The phosphonatebinds and sequesters at least ferrous iron, calcium and magnesiumcations. When bound to a sequestrant, these metal cations areunavailable to participate in the cellular metabolism of microorganisms,thus starving the microorganism of essential minerals. Further, there isincreasing evidence that the sequestrants serve to disrupt or compromisethe barrier properties of microbial cell membranes, thus allowingincreased permeability of membrane-soluble anti-microbial compounds.Membrane soluble anti-microbial compounds disrupt cellular physiology,such as the electron transfer system, and prevent the microorganism fromgrowing and reproducing. Many protonated weak acids and mostmono-terpenes are lipophilic and are able to disrupt cellularphysiology. The preservative properties of these substances are enhancedfurther by reducing the availability of potassium cation. Potassiumcation is required for active transport of one or more lipophilicanti-microbials out of the cell, so the microorganism is not able toexpel lipophilic weak acids and mono-terpenes, which build up and damageor kill the microorganism.

In certain exemplary embodiments, the beverage preservative system orthe beverage may further comprise a polyphosphate selected from thegroup consisting of sodium hexametaphosphate (SHMP), sodium acidmetaphosphate (SAMP), and a mixture thereof. Within the pH range of 2.5to 5.8, SAMP and SHMP can be substituted for one for the other in aratio of 1:1 without compromising their anti-microbial effect.Substitution of one for the other is often an issue of sensoryperception, particularly “mouthfeel”. Certain exemplary embodimentsinclude a polyphosphate at a concentration of about 1800 ppm or less(e.g., about 900 ppm or less, about 600 ppm or less).

The beverage preservative systems of invention are for use in high acidbeverages having a pH of about 5.8 or less. In certain exemplaryembodiments, the pH of the beverage preservative system or a beverageproduct comprising the preservative system is e.g., about pH 5.5 orless, about pH 4.6 or less, about pH 4.4 or less, about pH 2.9 to about4.4, about pH 2.5 to about 4.5, about pH 2.6 to about 3.8.

In general, the beverage preservative system or beverage product ofinvention should have a total concentration of chromium, aluminum,nickel, zinc, copper, manganese, cobalt, calcium, magnesium, and ironcations in the range of about 1.0 mM or less, e.g., about 0.5 mM to 0.75mM, about 0.54 mM or less. The present invention may optionally includeadded water that has been treated to remove metal cations. As opposed tothe teachings of U.S. Pat. No. 6,268,003, the preferred method oftreatment is via physical processes such as reverse osmosis and orelectro-deionization. Treatment by chemical means, as taught in U.S.Pat. No. 6,268,003 is acceptable, but is not preferred. The use ofchemical means to reduce water hardness often results in an increase inthe concentration of specific mono-valent cations, e.g., potassiumcations, that serve to compromise the invention described herein. Incertain exemplary embodiments, the added water has been treated byreverse osmosis, electro-deionization or both to decrease the totalconcentration of metal cations of chromium, aluminum, nickel, zinc,copper, manganese, cobalt, calcium, magnesium, and iron to about 1.0 mMor less.

As commonly understood in the art, the definitions of the termspreserve, preservative, and preservation do not provide a standard timeperiod for which the thing to be preserved is kept from spoilage,decomposition, or discoloration. The time period for “preservation” canvary greatly depending on the subject matter. Without a stated timeperiod, it can be difficult or impossible to infer the time periodrequired for a composition to act as a “preservative.”

Minimal inhibitory concentration (MIC) is another term for which nostandard time period is included in the definition. Typically, MICdescribes the concentration of a substance which measurably inhibits thegrowth of a single type of microorganism as compared to a positivecontrol without the substance. Any given MIC does not imply a specifictime period over which inhibition need occur. A substance may exhibit anobservable MIC during the first 24 hours of an experiment, but exhibitno measurable MIC relative to the positive control after 48 hours.

As used herein, the terms preserve, preservative, and preservation referto a food or beverage product protected against or a composition able toinhibit the growth of spoilage microorganisms for a period of at least16 weeks. Typically, the product is preserved under ambient conditions,which include the full range of temperatures experienced during storage,transport, and display (e.g., 0° C. to 40° C., 10° C. to 30° C., 20° C.to 25° C.) without limitation to the length of exposure to any giventemperature.

At least one biodegradable sequestrant (e.g.,ethylenediamine-N,N′-disuccinic acid (EDDS),ethylenediamine-N,N′-dimalonic acid (EDDM),ethylenediamine-N,N′-diglutaric acid (EDDG), or a combination of any ofthese), is present in certain exemplary embodiments of the beveragepreservative system or beverage product comprising the preservativesystem disclosed here. Each binds to a number of transition metalcations with binding affinity following the Irving-Williams series:Mn⁺²<Fe⁺²<Co⁺²<Ni⁺²<Cu⁺²>Zn⁺². Sufficient biodegradable sequestrant isincluded to bind cations of cobalt, chromium, copper, and nickel so asto lower the concentration of these metal cations enough to minimizebinding interactions with phosphonates. Certain exemplary embodiments ofthe present invention include biodegradable sequestrant at aconcentration in the range of about 15 ppm to about 500 ppm (e.g., about30 ppm to about 500 ppm, about 60 ppm to about 120 ppm, about 10 ppm toabout 120 ppm, about 10 ppm to about 75 ppm, about 10 ppm to about 45ppm, about 30 ppm to about 60 ppm, about 10 ppm to about 30 ppm). Thechemical structures of EDDS, EDDM, and EDDG are as follows:

Certain exemplary embodiments of the beverage preservative system or thebeverage product of invention include a sequestrant which binds cationsof chromium, aluminum, nickel, zinc, copper, manganese, and cobalt inpreference to calcium or magnesium cations. A non-exclusive example ofsuch a sequestrant is ethylenediamine tetraacetic acid (EDTA).Specifically, the various de-protonated species of the sequestrant whichcan bind metal cations must be present in a sufficient quantity (as afraction of the total sequestrant) to allow binding interaction witheach of the following metal cations: Co⁺², Cr⁺³, Al⁺³, Fe⁺³/Fe⁺², Cu⁺²,Ni⁺², Zn⁺², and Mn⁺². The pH of the solution dictates the proportions ofeach sequestrant species relative to the total concentration of thesequestrant. Each species of a sequestrant behaves similarly with regardto order of preference for the binding of metal cations, but differentspecies of a sequestrant will typical differ with regard to the amountof each metal that is bound at any particular pH. The maximum amount ofEDTA permitted in food and beverage products by the U.S. Food and DrugAdministration is 30 ppm. In certain exemplary embodiments, theconcentration of EDTA in finished beverage is about 75 ppm or less(e.g., about 45 ppm or less, about 30 ppm or less). Concentrates maycontain higher concentrations, as long as the concentration in finishedbeverage does not exceed legal limits. In certain exemplary embodiments,the beverage preservative system or the beverage product may compriseEDTA in order to stabilize certain chemical ingredients. When EDTA isincluded for this purpose, it may also secondarily act as an unintendedantimicrobial preservative. To stabilize chemical ingredients, EDTA isincluded in an amount no greater than 30 ppm. The chemical structure ofEDTA is as follows:

In certain exemplary embodiments, the beverage preservative system orbeverage product of invention includes one or more chemicals knowncollectively as “reverse sequestrants.” At least one class of reversesequestrants under consideration is natural in origin. Reversesequestrants differ from other sequestrants because of their ability totraverse lipid bi-layers such as found in the cell membrane of spoilagemicroorganisms. Reverse sequestrants chelate metal ions but have anoverall hydrophobic structure so that they tend to partition into thenon-aqueous phase of a water and oil mixture. For example, reversesequestrants tend to dissolve into the lipid membranes of microbes andtransport metal ions across the membrane. As a consequence, reversesequestrants are able compete with nuclear and cytoplasmic structuresfor the trace metal cations that have been absorbed by the spoilagemicroorganisms. A metal cation bound to reverse sequestrant will beunavailable for metabolic reactions, and this serves to reduce themicroorganism's ability to grow or to respond to damage by other typesof antimicrobial substances. Furthermore, a reverse sequestrant may alsotransport the trace metal cation to the cell exterior (by diffusion oractive expulsion) causing the metal cation to be lost to themicroorganism. Certain exemplary embodiments of the beveragepreservative systems or beverage products disclosed herein include atleast one reverse sequestrant at a concentration of about 150 ppm orless (e.g., about 100 ppm or less, about 75 ppm or less, about 60 ppm orless, about 30 ppm to about 100 ppm).

The antimicrobial effect of reverse sequestrants is increased byinclusion of a hydrophilic sequestrant in the beverage preservativesystem or beverage product. Non-exclusive examples are EDTA, EDDS, EDDM,EDDG, and mixtures of any of them. Such hydrophilic sequestrants aremembrane impermeable and so remain on the outside of microorganisms.However, these compounds are among the strongest sequestrants and willgenerally out-compete other types of sequestrants for any trace metalcations that are present. A reverse sequestrant binding a metal cationat the exterior surface of the cell membrane will be inclined to releasethe metal cation to the hydrophilic sequestrant. The extent and rate oftransfer of a trace metal cation from a reverse sequestrant (RS) to ahydrophilic sequestrant such as EDTA can be facilitated by employing an“effective” concentration of EDTA that is greater than that of thereverse sequestrant.

The cornerstone of this embodiment of the invention can be described bythe following process: (1) a reverse sequestrant binds to a trace metalcation on the cytosol side of the cell membrane of a microorganism, (2)the reverse sequestrant bound to the trace metal cation makes its wayacross the membrane to the cell exterior, (3) the trace metal cation isreleased by the reverse sequestrant at the exterior side of the cellmembrane and (4) becomes bound to a hydrophilic sequestrant such asEDTA, (5) the reverse sequestrant works its way back to the cytosol sideof the membrane and (6) initiates another round of metal cationtransport. This sequence of events is shown schematically in FIG. 1

The preservative effect of the reverse sequestrant and the hydrophilicsequestrant may be sufficient to prevent the outgrowth of spoilagemicroorganisms. At a minimum, they will make spoilage microorganismssignificantly more sensitive to other types of antimicrobials. It may bepreferable to incorporate into the beverage preservative system amembrane soluble antimicrobial agent such as a mono-terpene or a weakacid having an octanol/water partition coefficient Log P in the range of1.1 to 5.0. To the degree that the use of reverse sequestrants permitthe use of lowered concentrations of other preservative substances, thesensory impact of the beverage product is enhanced.

Various reverse sequestrants are contemplated for use in the presentinvention, some of which are natural compounds. Non-exclusive examplesare shown below and include:1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA),1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acidtetraacetoxymethyl ester (MAPTAM),N,N,N′,N′-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN), exochelin,pyridoxal isonicotinoyl hydrazone (PIH), 2-pyridylcarboxaldehydeisonicotinoyl hydrazone (PCIH), di-2-pyridylketone isonicotinoylhydrazone (PKIH), 2-quinolinecarboxaldehyde isonicotinoyl hydrazone(QCIH), 2-pyridylcarboxaldehyde 2-thiophenecarboxyl hydrazone (PCTH),di-2-pyridylketone 2-thiophenecarboxyl hydrazone (PKTH),2-quinolinecarboxaldehyde 2-thiophenecarboxyl hydrazone (QCTH),2-pyridylcarboxaldehyde m-bromobenzoyl hydrazone (PCBBH),2-pyridylcarboxaldehyde benzoyl hydrazone (PCBH), di-2-pyridylketonebenzoyl hydrazone (PKBH), 2-quinolinecarboxaldehyde benzoyl hydrazone(QCBH), 2-pyridylcarboxaldehyde p-aminobenzoyl hydrazone (PCAH),di-2-pyridylketone p-aminobenzoyl hydrazone (PKAH),2-quinolinecarboxaldehyde p-aminobenzoyl hydrazone (QCAH),2-pyridylcarboxaldehyde p-hydroxylbenzoyl hydrazone (PCHH),di-2-pyridylketone p-hydroxylbenzoyl hydrazone (PKHH),2-quinolinecarboxaldehyde p-hydroxylbenzoyl hydrazone (QCHH),2-furoylcarboxaldehyde isonicotinoyl hydrazone (FIH), and mixtures ofany of them.

Reverse Sequestrants

PCIH PKIH QCIH (2-pyridylcarboxaldehyde di 2-pyridylketone isonicotinoyl2-quinolinecarboxaldehyde isonicotinoyl hydrazone) hydrazoneisonicotinoyl hydrazone PCTH PKTH QCTH (2-pyridylcarboxaldehyde di2-pyridylketone thiophene 2-quinolinecarboxaldehyde thiophene hydrazone)hydrazone thiphene hydrazone PCBH PKBH QCBH (2-pyridylcarboxaldehyde di2-pyridylketone benzoyl 2-quinolinecarboxaldehyde benzoyl hydrazone)hydrazone benzoyl hydrazone PCAH PKAH QCAH (2-pyridylcarboxaldehydeamino di 2-pyridylketone amino benzoyl 2-quinolinecarboxaldehyde benzoylhydrazone) hydrazone amino benzoyl hydrazone PCHH PKHH QCHH(2-pyridylcarboxaldehyde di 2-pyridylketone hydroxy2-quinolinecarboxaldehyde hydroxy benzoyl hydrazone) benzoyl hydrazonehydroxy benzoyl hydrazone

R₁

PCIH PKIH QCIH

PCTH PKTH QCTH

PCBH PKBH QCBH

PCAH PKAH QCAH

PCHH PKHH QCHH

Certain exemplary embodiments of the beverage preservative system or thebeverage product of invention include at least one phosphonate whichtend to bind and sequester calcium and magnesium cations. Phosphonatesare organic compounds having at least one C—PO(OH)₂ group or C—PO(OR)₂group (with R═ alky, aryl). Phosphonates differ markedly from phosphatesin that phosphonates have a C—P bond whereas phosphates have only O—Pbonds. Phosphonates include phosphonic acids, their esters, and theirsalts, preferably their alkali metal salts. Bis-phosphonic acids (alsoknown as di-phosphonic acids) and their salts, preferably their alkalimetal salts, are a class of phosphonate having a P—C—P bond, asexemplified below:

Simple phosphonates (having a C—P bond) and bis-phosphonates (having aP—C—P bond) can be further categorized by the presence of nitrogen inthe molecule. For example, N-bis-phosphonates are metabolizeddifferently than non-nitrogenous bis-phosphonates.

The term N-bis-phosphonate includes primary to quaternary amines, andalso does not restrict the relative location of the nitrogen to theP—C—P bond within the molecule. A simple phosphonate containing anitrogen is referred to as an amino-phosphonate, and similarly includesprimary to quaternary amines and is not restricted to the relativelocation of the nitrogen to the C—P bond.

Phosphonates are not typically used as preservatives in food andbeverage products, and there is no known regulatory limit on the amountof phosphonate allowed in food and beverage products at the time of thiswriting. However, unlike polyphosphates, phosphonates are stable atambient and elevated temperatures in aqueous solution, and so can beused in thermally processed beverage products. Certain exemplaryembodiments of the beverage preservative system or beverage productcomprising the preservative system of invention include at least onephosphonate, preferably at a sufficient concentration to sequester andminimize the concentration of calcium and magnesium cations present inthe composition. Certain exemplary embodiments of the present inventioninclude phosphonate at two to three times the combined molarconcentration of the calcium and magnesium cations present. For example,phosphonate may be included at a concentration in the range of 1.06 mMto 1.59 mM, about 600 ppm to about 1000 ppm, or about 220 ppm to about540 ppm. To a degree, the ability of phosphonates to bind calcium andmagnesium cations is reduced in the presence of free copper or nickelcations, and so either minimization of copper and nickel cations or theaddition of another sequestrant to bind copper and nickel cations isimportant to the beverage preservation system.

Various phosphonates are contemplated for use in certain exemplaryembodiments of the present invention. In certain exemplary embodiments,phosphonates are included at a concentration of about 2100 ppm or less(e.g., about 1800 ppm or less, about 1500 ppm or less, about 1200 ppm orless, about 950 ppm or less, about 600 ppm or less, about 150 ppm orless). Non-exclusive examples of phosphonates are shown below andinclude: 2-hydroxyphosphonoacetic acid (HPAA), amino tri(methylenephosphonic acid), amino trimethylene pentasodium phosphonic acid,phenylphosphonic acid (PPA), methylphosphonic acid (MPA), 2-hydroxyphosphonoacetic acid (HPAA), 2-phosphonobutane-1,2,4-tricarboxylic acid,bis(hexamethylene triamine penta(methylenephosphonic acid)),ethylenediamine tetra(methylenephosphonic acid) (EDTMP), diethylenetriamine penta(methylenephosphonic acid),N-(phosphonoacetyl)-L-aspartate (PALA), N-cyclohexyliminodi(methylenephosphonic acid), N-iso-pentylimino di(methylenephosphonicacid), N-ethylimino di(methylenephosphonic acid), N-methyliminodi(methylenephosphonic acid), N-3-benzylimino di(methylenephosphonicacid), N-iso-pentylimino di(methylenephosphonic acid), N-3-picolyliminodi(methylenephosphonic acid), N-2-methyltetrahydrofuryliminodi(methylenephosphonic acid), and mixtures of any of them.

Phosphonate (Phosphonic Acids)

Various bis-phosphonates are contemplated for use in certain exemplaryembodiments of the present invention. In certain exemplary embodiments,bis-phosphonates are included at a concentration of about 1800 ppm orless (e.g., about 1500 ppm or less, about 1200 ppm or less, about 800ppm or less, about 500 ppm or less, about 125 ppm or less, about 600 ppmto about 1000 ppm, about 220 ppm to about 540 ppm). Non-exclusiveexamples of bis-phosphonates are shown below and include:

-   -   etidronate ((1-hydroxyethylidene)bis-phosphonate), clodronate,        methylene diphosphonic acid (MDPA), tiludronate        ([(4-chlorophenyl)thio]-methylene bis-phosphonate), tetraethyl        2-(3,5-di-tert-butyl-4-hydroxyphenyl)        ethenyl-1,1-bis-phosphonate, 1-hydroxyethane-1,1-diphosphonic        acid (HEDP), vinylidene-1,1-diphosphonic acid (VDP),        2-sulfonatoethylidene-1,1-diphosphonic acid (SEDP), and mixtures        of any of them.

Bis-Phosphonic Acids/Bis-Phosphonates

Various N-bis-phosphonic acids are contemplated for use in certainexemplary embodiments of the present invention. In certain exemplaryembodiments, N-bis-phosphonic acids are included at a concentration ofabout 1800 ppm or less (e.g., about 1500 ppm or less, about 1200 ppm orless, about 800 ppm or less, about 500 ppm or less, about 125 ppm orless). Non-exclusive examples of N-bis-phosphonic acids are shown belowand include: pamidronate, neridronate, olpadronate, risedronate,alendronate, zoledronate, ibandronate, incadronate, minodronate,piperidin-1-yl-methane-1,1-diphosphonic acid and derivatives thereofhaving at least one methyl or ethyl substituent on the piperidinyl ring,and mixtures of any of them.

N—Bis-Phosphonic Acids

Piperyd-1-yl-methane-1,1 diphosphonic Acid Derivatives

Certain exemplary embodiments of the present invention include amono-terpene and/or a weak acid, each having an octanol/water partitioncoefficient Log P in the range of 1.1 to 5.0, which has been found todisrupt cellular function as assayed by methods such as flow cytometry.The weak acid should predominantly exist in its protonated form at pHbelow 4. Non-limiting examples of such weak acids are cinnamic acid(e.g., trans-cinnamic acid) and sorbic acid. Additionally, esters ofhydroxybenzoic acid may be included. When included with sequestrants inthe beverage preservative systems and beverage products of invention,lower than expected concentrations of mono-terpene and/or weak acid arerequired for a preservative effect. It is believed that metal cationswhich are bound by sequestrants are then unavailable to degrade themono-terpenes and/or weak acids, and render the cell membranes ofmicroorganisms more permeable to these anti-microbial compounds. Certainexemplary embodiments of the present invention include the mono-terpene,the weak acid, or a mixture thereof at a concentration in the range ofabout 500 ppm or less, e.g., about 150 ppm or less, about 25 ppm toabout 200 ppm. Certain exemplary embodiments of the present inventioninclude trans-cinnamic acid at a concentration in the range of about 50ppm to about 150 ppm. Certain exemplary embodiments of the presentinvention include sorbic acid at a concentration in the range of about500 ppm to about 800 ppm. Certain exemplary embodiments include a weakacid at a concentration in the range of about 10 ppm to about 850 ppm(e.g., about 10 ppm to about 650 ppm, about 100 ppm to about 500 ppm,about 500 ppm to about 800 ppm, about 250 ppm to about 500 pm, about 100ppm to about 250 ppm, about 10 ppm to about 50 ppm).

Certain exemplary embodiments of the beverage preservative system orbeverage product of invention include a surfactant such as lauricarginate. The surfactant compromises the outer protective barrier of thespoilage microorganism, thus rendering its cell membrane more permeableto the above preservative compounds. In certain exemplary embodiments,lauric arginate is included at a concentration in the range of about 2.5ppm to about 25 ppm. The chemical structure of lauric arginate is asfollows:

Certain exemplary embodiments of the beverage preservative system orbeverage product of invention have minimal levels of potassium cation. Alack of potassium cations prevents microorganisms from activelyexpelling preservatives such as mono-terpenes, weak acids, and esters ofhydroxybenzoic acid, thus enhancing the anti-microbial effect of thesepreservatives. This factor is one of the reasons why it is preferredthat treatment of added water should not include chemical methods suchas ion-exchange that can lead to increased concentration of potassiumion. In certain exemplary embodiments, the concentration of potassiumion is about 150 mM or less, e.g. about 75 ppm or less, about 15 mM orless, about 15 ppm or less.

Beverage products according to the present invention include both stilland carbonated beverages wherein the beverage pH is in the range of 5.8or below (e.g., about pH 5.5 or less, about pH 4.6 or less, about pH 4.4or less, about pH 2.9 to about 4.4, about pH 2.5 to about 4.5, about pH2.6 to about 3.8). Herein, the term carbonated beverage is inclusive ofany combination of water, juice, flavor and sweetener that is meant tobe consumed as an alcohol free liquid and which also is made to possessa carbon dioxide concentration of 0.2 volumes of CO₂ or greater. Theterm “volume of CO₂” is understood to mean a quantity of carbon dioxideabsorbed into the liquid wherein one volume CO₂ is equal to 1.96 gramsof carbon dioxide (CO₂) per liter of product (0.0455M) at 25° C.Non-inclusive examples of carbonated beverages include flavored seltzerwaters, juices, cola, lemon-lime, ginger ale, and root beer beverageswhich are carbonated in the manner of soft drinks, as well as beveragesthat provide health or wellness benefits from the presence ofmetabolically active substances, such as vitamins, amino acids,proteins, carbohydrates, lipids, or polymers thereof. Such products mayalso be formulated to contain milk, coffee, or tea or other botanicalsolids. It is also possible to formulate such beverages to contain oneor more nutraceuticals. Herein, a nutraceutical is a substance that hasbeen shown to possess, minimally, either a general or specific healthbenefit or sense of wellness as documented in refereed professionaljournals or texts. Nutraceuticals do not necessarily act to either cureor prevent specific types of medical conditions.

Herein, the term “still beverage” is any combination of water andingredient which is meant to be consumed in the manner of an alcoholfree liquid beverage and which possess no greater than 0.2 volumescarbon dioxide. Non-inclusive examples of still beverages includeflavored waters, tea, coffee, nectars, mineral drinks, sports beverages,vitamin waters, juice-containing beverages, punches or the concentratedforms of these beverages, as well as beverage concentrates which containat least about 45% by weight of juice. Such beverages may besupplemented with vitamins, amino acids, protein-based,carbohydrate-based or lipid-based substances. As noted, the inventionincludes juice containing products, whether carbonated or still. “Juicecontaining beverages” or “Juice beverages”, regardless of whether stillor carbonated, are products containing some or all the components of afruit, vegetable or nuts or mixture thereof that can either be suspendedor made soluble in the natural liquid fraction of the fruit.

The term vegetable, when used herein, is inclusive both fruiting and thenon-fruit but edible portion of plants such as tubers, leaves, rinds,and also, if not otherwise indicated, any grains, nuts, beans, andsprouts which are provided as juices or beverage flavorings. Unlessdictated by local, national regional regulatory agencies the selectiveremoval of certain substances (pulp, pectins, etc) does not constitutean adulteration of a juice.

By way of example, juice products and juice drinks can be obtained fromthe fruit of apple, cranberry, pear, peach, plum, apricot, nectarine,grape, cherry, currant, raspberry, goose-berry, blackberry, blueberry,strawberry, lemon, orange, grapefruit, passionfruit, mandarin,mirabelle, tomato, lettuce, celery, spinach, cabbage, watercress,dandelion, rhubarb, carrot, beet, cucumber, pineapple, custard-apple,coconut, pomegranate, guava, kiwi, mango, papaya, watermelon, lo hanguo, cantaloupe, pineapple, banana or banana puree, lemon, tomato,mango, papaya, lime, tangerine, cherry, raspberry, carrot and mixturesthereof. Preferred juices are the citrus juices, and most preferred arethe non-citrus juices, apple, pear, cranberry, strawberry, grape,papaya, mango and cherry.

Not all ranges of juice concentration can be employed. The inventioncould be used to preserve a formulation that is essentially 100% juiceif then the presence of specific metal cation species are not exceeded.Another possibility would be to treat the juice in such a manner as tolower the concentration of specific metal cation species. Similar issuesarise for juice beverages, which typically contain at least 95% juice.Formulations containing juice concentrations as high as 10% may bepreserved by this invention and certainly. A beverage containing lessthan 10% juice would be very likely preserved by this invention abeverage containing no more than 5% juice would be preserved by thisinvention. Any juice can be used to make the beverage of this invention.If a beverage concentrate is desired, the fruit juice is concentrated byconventional means to from about 20° Brix to about 80° Brix. Beverageconcentrates are usually 40° Brix or higher (about 40% to about 75%sugar solids).

Typically, beverages will possess a specified range of acidity. Acidityof a beverage is largely determined by the type of acidulant, itsconcentration, and the propensity of protons associated with the acid todissociate away from the acid when the acid is entered into solution.Any solution with a measurable pH between 0-14 possesses some measure ofacidity. However, those solutions with pH below 7 are generallyunderstood to be acidic and those above pH 7 are understood to be basic.The acidulant can be organic or inorganic. Non-exclusive examples oforganic acids are citric, malic, ascorbic, tartaric, lactic, gluconic,and succinic acids. Non-exclusive examples of inorganic acids are thephosphoric acid compounds and the mono- and di-potassium salts of theseacids. (Mono- and di-potassium salts of phosphoric acid possess at leastone proton that can contribute to acidity).

The various acids can be combined with salts of the same or differentacids in order to manage pH or the buffer capacity of the beverage to aspecified pH or range of pH. The invention can function at a pH as lowas 2.6, but the invention will function best as the pH is increased from2.6 up to pH 3.8. The invention is not limited by the type of acidulantemployed in acidifying the product as long as the final pH of theproduct does not exceed pH 5.8, preferably not exceeding pH 4.5.Virtually any organic acid salt can be used so long as it is edible anddoes not provide an off-flavor. The choice of salt or salt mixture willbe determined by the solubility and the taste. Citrate, malate andascorbate yield ingestible complexes whose flavors are judged to bequite acceptable, particularly in fruit juice beverages. Tartaric acidis acceptable, particularly in grape juice beverages, as is lactic acid.Longer-chain fatty acids may be used but can affect flavor and watersolubility. For essentially all purposes, the malate, gluconate, citrateand ascorbate moieties suffice.

Certain exemplary embodiments of the beverage product of inventioninclude sports (electrolyte balancing) beverages (carbonated ornon-carbonated). Typical sport beverages contain water, sucrose syrup,glucose-fructose syrup, and natural or artificial flavors. Thesebeverages can also contain sodium chloride, citric acid, sodium citrate,mono-potassium phosphate, as well as other natural or artificialsubstances which serve to replenish the balance of electrolytes lostduring perspiration.

In certain exemplary embodiments, the present invention is also includesbeverage formulations supplemented with fat soluble vitamins.Non-exclusive examples of vitamins include fat-soluble vitamin E or itsesters, vitamin A or its esters, vitamin K, and vitamin D3, especiallyvitamin E and vitamin E acetate. The form of the supplement can bepowder, gel or liquid or combination thereof. Fat-soluble vitamins maybe added in a restorative amount, i.e. enough to replace vitaminnaturally present in a beverage such as juice or milk, which may havebeen lost or inactivated during processing. Fat-soluble vitamins mayalso be added in a nutritionally supplemental amount, i.e. an amount ofvitamin considered advisable for a child or adult to consume based onRDAs and other such standards, preferably from about one to three timesthe RDA (Recommended Daily Amount). Other vitamins which can be added tothe beverages include vitamin B niacin, pantothenic acid, folic acid,vitamin D, vitamin E, vitamin B and thiamine. These vitamins be added atlevels of from 10% to 300% RDA. It need be recognized that a potentialexists for some types of guest molecules or complexes to becomeentrapped into certain types of micelles, liposomes, or fat globules butthis can only be characterized on a case by case basis.

Supplements: The invention can be compromised by the presence of certaintypes of supplements but it is not an absolute and it will vary frombeverage formulation to beverage formulation. The degree to which theinvention is compromised will depend on the nature of the supplement andthe resulting concentration of specific metal cations in the beverage asa consequence of the presence of the supplement. For example, calciumsupplements can compromise the invention, but not to the same degree aschromium supplements. Calcium supplements may be added to the degreethat a critical value total calcium concentration is not exceeded (e.g.,⅓ to ½ the molar concentration of diphosphonic acid in the beverage).Calcium sources that are compatible with the invention include calciumorganic acid complexes. Among the preferred calcium sources is “calciumcitrate-malate”, as described in U.S. Pat. No. 4,786,510 and U.S. Pat.No. 4,786,518 issued to Nakel et al. (1988) and U.S. Pat. No. 4,722,847issued to Heckert (1988). Other calcium sources compatible with theinvention include calcium acetate, calcium tartrate, calcium lactate,calcium malate, calcium citrate, calcium phosphate, calcium orotate, andmixtures thereof. Calcium chloride and calcium sulfate can also beincluded; however at higher levels they taste astringent.

Flavor Component: Beverage products according to the present inventioncan contain flavors of any type. The flavor component of the presentinvention contains flavors selected from artificial, natural flavors,botanical flavors fruit flavors and mixtures thereof. The term“botanical flavor” refers to flavors derived from parts of a plant otherthan the fruit; i.e. derived from bean, nuts, bark, roots and leaves.Also included within the term “botanical flavor” are syntheticallyprepared flavors made to simulate botanical flavors derived from naturalsources. Examples of such flavors include cocoa, chocolate, vanilla,coffee, kola, tea, and the like. Botanical flavors can be derived fromnatural sources such as essential oils and extracts, or can besynthetically prepared. The term “fruit flavors” refers to those flavorsderived from the edible reproductive part of a seed plant, especiallyone having a sweet pulp associated with the seed. Also included withinthe term “fruit flavor” are synthetically prepared flavors made tosimulate fruit flavors derived from natural sources.

Artificial flavors can also be employed. Non-exclusive examples ofartificial flavors include chocolate, strawberry, vanilla, cola, orartificial flavors that mimic a natural flavor can be used to formulatea still or carbonated beverage flavored to taste like fruit. Theparticular amount of the flavor component effective for imparting flavorcharacteristics to the beverage mixes of the present invention (“flavorenhancing”) can depend upon the flavor(s) selected, the flavorimpression desired, and the form of the flavor component. The flavorcomponent can comprise at least 0.005% by weight of the beveragecomposition.

On a case by case basis, the beverage preservative system according tothe present invention is compatible with beverages formulated to containaqueous essence. As used herein, the term “aqueous essence” refers tothe water soluble aroma and flavor materials which are derived from,fruit juices. Aqueous essences can be fractionated, concentrated orfolded essences, or enriched with added components. As used herein, theterm “essence oil” refers to the oil or water insoluble fraction of thearoma and flavor volatiles obtained from juices. Orange essence oil isthe oily fraction which separates from the aqueous essence obtained byevaporation of orange juice. Essence oil can be fractionated,concentrated or enriched. As used herein, the term “peel oil” refers tothe aroma and flavor derived from oranges and other citrus fruit islargely composed of terpene hydrocarbons, e.g. aliphatic aldehydes andketones, oxygenated terpenes and sesquiterpenes. From about 0.002% toabout 1.0% of aqueous essence and essence oil are used in citrusflavored juices.

Sweetener Component: The present invention is not affected by the typeor concentration of sweeteners, wherein in sweetener is among thosecommonly employed for use in beverages. The sweetener can include amonosaccharide or a disaccharide. A certain degree of purity fromcontamination by metal cations will be expected. Peptides possessingsweet taste are also permitted. The most commonly employed saccharidesinclude sucrose, fructose, dextrose, maltose and lactose and invertsugar. Mixtures of these sugars can be used. Other natural carbohydratescan be used if less or more sweetness is desired. Other types of naturalsweeteners structured from carbon, hydrogen and oxygen, e.g.,rebaudioside A, stevioside, Lo Han Guo, mogroside V, monatin, can alsobe used. The present invention is also compatible with artificialsweeteners. By way of example, artificial sweeteners include saccharin,cyclamates, acetosulfam, mogroside, Laspartyl-L-phenylalanine loweralkyl ester sweeteners (e.g. aspartame), L-aspartyl-D-alanine amides asdisclosed in U.S. Pat. No. 4,411,925 to Brennan et al. (1983),L-aspartyl-D-serine amides as disclosed in U.S. Pat. No. 4,399,163 toBrennan et al., (1983), L-aspartyl-L-lhydroxymethyl alkaneamidesweeteners as disclosed in U.S. Pat. No. 4,338,346 to Brand, issued Dec.21, 1982, L-aspartyl-1-hydroxy ethylakaneamide sweeteners as disclosedin U.S. Pat. No. 4,423,029 to Rizzi, (1983), L-aspartyl-D-phenylglycineester and amide sweeteners as disclosed in European Patent Application168,112 to J. M. Janusz, published Jan. 15, 1986, and the like. Aparticularly preferred sweetener is aspartame. The amount of thesweetener effective in the beverage mixes of the invention depends uponthe particular sweetener used and the sweetness intensity desired.

Head space atmosphere: The presence of either air in the headspace ofthe beverage product will have no measurable impact on the compositionof the invention. The presence of carbon dioxide gas or other gases thatcause the exclusion of oxygen from the beverage (nitrogen, nitrousoxide, etc) may permit the use of reduced concentrations of chemicalpreservatives employed along with the sequestrants. The concentration ofsequestrants required will be dictated only by the type and amount ofmetal cations that are present in the beverage product.

The following example is a specific embodiment of the present invention,but is not intended to limit it. Any patent document referenced hereinis incorporated in its entirety for all purposes.

Example 1

According to one embodiment of the invention, a beverage preservativesystem is provided which comprises:

-   -   a. sorbic acid, cinnamic acid, alkali metal salts thereof (e.g.,        K⁺, Na⁺), or mixtures of any of them that result in specific        concentrations of cinnamic or sorbic acid as determined by final        beverage pH;    -   b. a bio-degradable sequestrant selected from the group        consisting of ethylenediamine-N,N′-disuccinic acid (EDDS),        ethylenediamine-N,N′-dimalonic acid (EDDM),        ethylenediamine-N,N′-diglutaric acid (EDDG), or mixtures of any        of them, and    -   c. a pH of 5.8 or less;    -   d. wherein the beverage preservative system prevents spoilage by        microorganisms in a beverage within a sealed container for a        period of at least 16 weeks.

In some instances, EDTA may need to be present where the purpose is tostabilize chemical ingredients. When it is added for this purpose, itwill fulfill the un-intended second role should it participate as anun-intended antimicrobial preservative. To fulfill the role ofstabilization of chemical ingredients, EDTA need not be present in anamount greater than 30 ppm.

Assuming compliance to upper limit constraints for the concentration ofCopper, Cobalt, Iron, Zinc, Aluminum, Chromium, and Nickel, the amountof preservatives listed in Example 1 that are required to stabilize abeverage are found to be dictated by the combined effect of two factors;the concentration of Calcium and the concentration of AssimilableNitrogen. Assimilable Nitrogen is principally an estimate of the reducedform of nitrogen (NH and NH₂) available in solution and serves as aestimate for the availability of those forms of nitrogen (ammonia, urea,amino acids and peptides) that can be employed as nutrients by thespoilage microorganism. A measure of total nitrogen would include allforms of nitrogen, some (Nitrate and Nitrite) which are not assimilableby fungi and many types of spoilage bacteria. Exemplary ranges for theconcentration of biodegradable sequestrant and weak acid for Ex. 1 areshown below.

Formulation Requirements 0.26 mM < [Ca++] < 0.43 mM < [Ca++] < Example 1[Ca++] < 0.26 mM 0.43 mM 0.764M AN < 3 ppm 10 ppm < [WA] < 1 ppm < [WA]< 50 ppm 1 ppm < [WA] < 50 ppm 10 ppm < 30 ppm < [BDS] < 50 ppm [BDS] <30 ppm 60 ppm 60 ppm < [BDS] < 120 ppm 3 ppm < AN < 50 ppm < [WA] < 100ppm < [WA] < 250 ppm < [WA] < 10 ppm 50 ppm 10 ppm < 250 ppm 500 ppm[BDS] < 30 ppm 30 ppm < [BDS] < 60 ppm < [BDS] < 60 ppm 120 ppm 10 ppm <AN < 100 ppm < [WA] < 250 ppm < [WA] < 500 ppm < [WA] < 20 ppm 250 ppm10 ppm < 500 ppm 800 ppm [BDS] < 30 ppm 30 ppm < [BDS] < 60 ppm < [BDS]< 60 ppm 120 ppm WA = weak acid BDS = bio degradable sequestrant AN =Assimilable Nitrogen

In the following examples, in some instances, EDTA may need to bepresent where the purpose is to stabilize chemical ingredients. When itis added for this purpose, it will fulfill the un-intended second roleshould it participate as an un-intended antimicrobial preservative. Tofulfill the role of stabilization of chemical ingredients, EDTA need notbe present in an amount greater than 30 ppm.

In the following examples which utilize SAMP and SHMP, within the pHrange of 2.5 to 5.8, SAMP and SHMP can substitute for one another in aratio of 1:1 without compromising the anti-microbial effect.Substitution of one for other is often an issue of sensory perception,particularly “mouthfeel”.

In the following examples which utilize phosphonic acids orphosphonates, phosphonic acids disclosed above can substitute for oneanother in a ratio of 1:1 without compromise to anti-microbial effect.Substitution of one for other is often an issue of sensory perception,particularly “mouth feel”. It is anticipated that any currently knownmonomer phosphonate will function similar to those disclosed above.

In the following examples which utilize bis-phosphonates, the basicstructure and description of bis-phosphonates are disclosed above.Structures above can substitute for one another in a ratio of 1:1without compromise to anti-microbial effect. Substitution of one forother is often an issue of sensory perception, particularly “mouthfeel”. It is anticipated that any currently known monomerbis-phosphonate will function similar to those found above with regardto antimicrobial effect.

In the following examples which utilize N-bis-phosphonates, the basicstructure and description of N-bis-phosphonate is disclosed above.Structures above can substitute for one another in a ratio of 1:1without compromise to anti-microbial effect. Substitution of one forother is often an issue of sensory perception, particularly “mouthfeel”. It is anticipated that any currently known monomerbis-phosphonate will function similar to those found above with regardto antimicrobial effect.

In the following examples that utilize reverse sequestrants, the basicstructure and description of reverse sequestrants is disclosed above.Structures above can substitute for one another in a ratio of 1:1without compromise to anti-microbial effect. Substitution of one forother is often an issue of sensory perception, particularly “mouthfeel”. It is anticipated that any currently known reverse sequestrantwill function similar to those found above with regard to antimicrobialeffect. Reverse sequestrants possess demonstrated hydrophobic structureand a tendency to partition in the non-aqueous phase of a water and oilmixture.

Example 2

According to another embodiment of the invention, a beveragepreservative system is provided which comprises:

-   -   a. sorbic acid, cinnamic acid, alkali metal salts thereof (e.g.,        K⁺, Na⁺), or mixtures of any of them that result in specific        concentrations of cinnamic or sorbic acid as determined by final        beverage pH;    -   b. a bio-degradable sequestrant selected from the group        consisting of ethylenediamine-N,N′-disuccinic acid (EDDS),        ethylenediamine-N,N′-dimalonic acid (EDDM),        ethylenediamine-N,N′-diglutaric acid (EDDG), or mixtures of any        of them, and    -   c. a pH of 5.8 or less;    -   d. wherein the beverage preservative system prevents spoilage by        microorganisms in a beverage within a sealed container for a        period of at least 16 weeks.

EDTA may need to be present where the purpose is to stabilize chemicalingredients.

The beverage preservative system also comprises Sodium Hexametaphosphate(SHMP), Sodium Acid Metaphosphate (SAMP), or mixture of SHMP and SAMP toa prescribed total amount.

Example 3

According to another embodiment of the invention, a beveragepreservative system is provided which comprises:

-   -   a. sorbic acid, cinnamic acid, alkali metal salts thereof (e.g.,        K⁺, Na⁺), or mixtures of any of them that result in specific        concentrations of cinnamic or sorbic acid as determined by final        beverage pH;    -   b. a bio-degradable sequestrant selected from the group        consisting of ethylenediamine-N,N′-disuccinic acid (EDDS),        ethylenediamine-N,N′-dimalonic acid (EDDM),        ethylenediamine-N,N′-diglutaric acid (EDDG), or mixtures of any        of them, and    -   c. a pH of 5.8 or less;    -   d. wherein the beverage preservative system prevents spoilage by        microorganisms in a beverage within a sealed container for a        period of at least 16 weeks.

EDTA may need to be present where the purpose is to stabilize chemicalingredients.

The beverage preservative system also comprises a phosphonate orphosphonic acid to a prescribed total amount wherein the compositioncontains any number of types of phosphonate structures such that a totalamount of phosphonate is achieved.

Example 4

According to another embodiment of the invention, a beveragepreservative system is provided which comprises:

-   -   a. sorbic acid, cinnamic acid, alkali metal salts thereof (e.g.,        K⁺, Na⁺), or mixtures of any of them that result in specific        concentrations of cinnamic or sorbic acid as determined by final        beverage pH;    -   b. a bio-degradable sequestrant selected from the group        consisting of ethylenediamine-N,N′-disuccinic acid (EDDS),        ethylenediamine-N,N′-dimalonic acid (EDDM),        ethylenediamine-N,N′-diglutaric acid (EDDG), or mixtures of any        of them, and    -   c. a pH of 5.8 or less;    -   d. wherein the beverage preservative system prevents spoilage by        microorganisms in a beverage within a sealed container for a        period of at least 16 weeks.

EDTA may need to be present where the purpose is to stabilize chemicalingredients.

The beverage preservative system also comprises a phosphonate orphosphonic acid to a prescribed total amount wherein the compositioncontains any number of types of phosphonate structures such that a totalamount of phosphonate is achieved.

The beverage preservative system also comprises Sodium Hexametaphosphate(SHMP) Sodium Acid Metaphosphate (SAMP), or mixture of SHMP and SAMP toa prescribed total amount.

Example 5

According to another embodiment of the invention, a beveragepreservative system is provided which comprises:

-   -   a. sorbic acid, cinnamic acid, alkali metal salts thereof (e.g.,        K⁺, Na⁺), or mixtures of any of them that result in specific        concentrations of cinnamic or sorbic acid as determined by final        beverage pH;    -   b. a bio-degradable sequestrant selected from the group        consisting of ethylenediamine-N,N′-disuccinic acid (EDDS),        ethylenediamine-N,N′-dimalonic acid (EDDM),        ethylenediamine-N,N′-diglutaric acid (EDDG), or mixtures of any        of them, and    -   c. a pH of 5.8 or less;    -   d. wherein the beverage preservative system prevents spoilage by        microorganisms in a beverage within a sealed container for a        period of at least 16 weeks.

EDTA may need to be present where the purpose is to stabilize chemicalingredients.

The beverage preservative system also comprises a phosphonate orphosphonic acid to a prescribed total amount wherein the compositioncontains any number of types of phosphonate structures such that a totalamount of phosphonate is achieved.

The beverage preservative system also comprises Sodium Hexametaphosphate(SHMP) Sodium Acid Metaphosphate (SAMP), or mixture of SHMP and SAMP toa prescribed total amount.

Example 6

According to another embodiment of the invention, a beveragepreservative system is provided which comprises:

-   -   a. sorbic acid, cinnamic acid, alkali metal salts thereof (e.g.,        K⁺, Na⁺), or mixtures of any of them that result in specific        concentrations of cinnamic or sorbic acid as determined by final        beverage pH;    -   b. a bio-degradable sequestrant selected from the group        consisting of ethylenediamine-N,N′-disuccinic acid (EDDS),        ethylenediamine-N,N′-dimalonic acid (EDDM),        ethylenediamine-N,N′-diglutaric acid (EDDG), or mixtures of any        of them, and    -   c. a pH of 5.8 or less;    -   d. wherein the beverage preservative system prevents spoilage by        microorganisms in a beverage within a sealed container for a        period of at least 16 weeks.

The beverage preservative system also comprises a bis-phosphonate to aprescribed total amount wherein the composition contains any number oftypes of bis-phosphonate structures such that a total amount ofbis-phosphonate is achieved.

The beverage preservative system also comprises a phosphonate orphosphonic acid to a prescribed total amount wherein the compositioncontains any number of types of phosphonate structures such that a totalamount of phosphonate is achieved.

Example 7

According to another embodiment of the invention, a beveragepreservative system is provided which comprises:

-   -   a. sorbic acid, cinnamic acid, alkali metal salts thereof (e.g.,        K⁺, Na⁺), or mixtures of any of them that result in specific        concentrations of cinnamic or sorbic acid as determined by final        beverage pH;    -   b. a bio-degradable sequestrant selected from the group        consisting of ethylenediamine-N,N′-disuccinic acid (EDDS),        ethylenediamine-N,N′-dimalonic acid (EDDM),        ethylenediamine-N,N′-diglutaric acid (EDDG), or mixtures of any        of them, and    -   c. a pH of 5.8 or less;    -   d. wherein the beverage preservative system prevents spoilage by        microorganisms in a beverage within a sealed container for a        period of at least 16 weeks.

EDTA may need to be present where the purpose is to stabilize chemicalingredients.

The beverage preservative system also comprises a bis-phosphonate to aprescribed total amount wherein the composition contains any number oftypes of bis-phosphonate structures such that a total amount ofbis-phosphonate is achieved.

The beverage preservative system also comprises a phosphonate orphosphonic acid to a prescribed total amount wherein the compositioncontains any number of types of phosphonate structures such that a totalamount of phosphonate is achieved.

The beverage preservative system also comprises Sodium Hexametaphosphate(SHMP) Sodium Acid Metaphosphate (SAMP), or mixture of SHMP and SAMP toa prescribed total amount.

Example 8

According to another embodiment of the invention, a beveragepreservative system is provided which comprises:

-   -   a. sorbic acid, cinnamic acid, alkali metal salts thereof (e.g.,        K⁺, Na⁺), or mixtures of any of them that result in specific        concentrations of cinnamic or sorbic acid as determined by final        beverage pH;    -   b. a bio-degradable sequestrant selected from the group        consisting of ethylenediamine-N,N′-disuccinic acid (EDDS),        ethylenediamine-N,N′-dimalonic acid (EDDM),        ethylenediamine-N,N′-diglutaric acid (EDDG), or mixtures of any        of them, and    -   c. a pH of 5.8 or less;    -   d. wherein the beverage preservative system prevents spoilage by        microorganisms in a beverage within a sealed container for a        period of at least 16 weeks.

The beverage preservative system also comprises a bis-phosphonate to aprescribed total amount wherein the composition contains any number oftypes of bis-phosphonate structures such that a total amount ofbis-phosphonate is achieved.

The beverage preservative system also comprises a phosphonate orphosphonic acid to a prescribed total amount wherein the compositioncontains any number of types of phosphonate structures such that a totalamount of phosphonate is achieved.

The beverage preservative system also comprises Sodium Hexametaphosphate(SHMP) Sodium Acid Metaphosphate (SAMP), or mixture of SHMP and SAMP toa prescribed total amount.

Example 9

According to another embodiment of the invention, a beveragepreservative system is provided which comprises:

-   -   a. sorbic acid, cinnamic acid, alkali metal salts thereof (e.g.,        K⁺, Na⁺), or mixtures of any of them that result in specific        concentrations of cinnamic or sorbic acid as determined by final        beverage pH;    -   b. a bio-degradable sequestrant selected from the group        consisting of ethylenediamine-N,N′-disuccinic acid (EDDS),        ethylenediamine-N,N′-dimalonic acid (EDDM),        ethylenediamine-N,N′-diglutaric acid (EDDG), or mixtures of any        of them, and    -   c. a pH of 5.8 or less;    -   d. wherein the beverage preservative system prevents spoilage by        microorganisms in a beverage within a sealed container for a        period of at least 16 weeks.

EDTA may need to be present where the purpose is to stabilize chemicalingredients.

The beverage preservative system also comprises an N-bis-phosphonate toa prescribed total amount wherein the composition contains any number oftypes of N-bis-phosphonate structures such that a total amount ofbis-phosphonate is achieved.

Example 10

According to another embodiment of the invention, a beveragepreservative system is provided which comprises:

-   -   a. sorbic acid, cinnamic acid, alkali metal salts thereof (e.g.,        K⁺, Na⁺), or mixtures of any of them that result in specific        concentrations of cinnamic or sorbic acid as determined by final        beverage pH;    -   b. a bio-degradable sequestrant selected from the group        consisting of ethylenediamine-N,N′-disuccinic acid (EDDS),        ethylenediamine-N,N′-dimalonic acid (EDDM),        ethylenediamine-N,N′-diglutaric acid (EDDG), or mixtures of any        of them, and    -   c. a pH of 5.8 or less;    -   d. wherein the beverage preservative system prevents spoilage by        microorganisms in a beverage within a sealed container for a        period of at least 16 weeks.

EDTA may need to be present where the purpose is to stabilize chemicalingredients.

The beverage preservative system also comprises an N-bis-phosphonate toa prescribed total amount wherein the composition contains any number oftypes of N-bis-phosphonate structures such that a total amount ofbis-phosphonate is achieved.

The beverage preservative system also comprises a bis-phosphonate to aprescribed total amount wherein the composition contains any number oftypes of bis-phosphonate structures such that a total amount ofbis-phosphonate is achieved.

Example 11

According to another embodiment of the invention, a beveragepreservative system is provided which comprises:

-   -   a. sorbic acid, cinnamic acid, alkali metal salts thereof (e.g.,        K⁺, Na⁺), or mixtures of any of them that result in specific        concentrations of cinnamic or sorbic acid as determined by final        beverage pH;    -   b. a bio-degradable sequestrant selected from the group        consisting of ethylenediamine-N,N′-disuccinic acid (EDDS),        ethylenediamine-N,N′-dimalonic acid (EDDM),        ethylenediamine-N,N′-diglutaric acid (EDDG), or mixtures of any        of them, and    -   c. a pH of 5.8 or less;    -   d. wherein the beverage preservative system prevents spoilage by        microorganisms in a beverage within a sealed container for a        period of at least 16 weeks.

EDTA may need to be present where the purpose is to stabilize chemicalingredients.

The beverage preservative system also comprises an N-bis-phosphonate toa prescribed total amount wherein the composition contains any number oftypes of N-bis-phosphonate structures such that a total amount ofbis-phosphonate is achieved.

The beverage preservative system also comprises a phosphonate orphosphonic acid to a prescribed total amount wherein the compositioncontains any number of types of phosphonate structures such that a totalamount of phosphonate is achieved.

Example 12

According to another embodiment of the invention, a beveragepreservative system is provided which comprises:

-   -   a. sorbic acid, cinnamic acid, alkali metal salts thereof (e.g.,        K⁺, Na⁺), or mixtures of any of them that result in specific        concentrations of cinnamic or sorbic acid as determined by final        beverage pH;    -   b. a bio-degradable sequestrant selected from the group        consisting of ethylenediamine-N,N′-disuccinic acid (EDDS),        ethylenediamine-N,N′-dimalonic acid (EDDM),        ethylenediamine-N,N′-diglutaric acid (EDDG), or mixtures of any        of them, and    -   c. a pH of 5.8 or less;    -   d. wherein the beverage preservative system prevents spoilage by        microorganisms in a beverage within a sealed container for a        period of at least 16 weeks.

EDTA may need to be present where the purpose is to stabilize chemicalingredients.

The beverage preservative system also comprises an N-bis-phosphonate toa prescribed total amount wherein the composition contains any number oftypes of N-bis-phosphonate structures such that a total amount ofbis-phosphonate is achieved.

The beverage preservative system also comprises a phosphonate or aphosphonic acid to a prescribed total amount wherein the compositioncontains any number of types of phosphonate structures such that a totalamount of phosphonate is achieved.

The beverage preservative system also comprises Sodium Hexametaphosphate(SHMP) Sodium Acid Metaphosphate (SAMP), or mixture of SHMP and SAMP toa prescribed total amount.

Example 13

According to another embodiment of the invention, a beveragepreservative system is provided which comprises:

-   -   a. sorbic acid, cinnamic acid, alkali metal salts thereof (e.g.,        K⁺, Na⁺), or mixtures of any of them that result in specific        concentrations of cinnamic or sorbic acid as determined by final        beverage pH;    -   b. a bio-degradable sequestrant selected from the group        consisting of ethylenediamine-N,N′-disuccinic acid (EDDS),        ethylenediamine-N,N′-dimalonic acid (EDDM),        ethylenediamine-N,N′-diglutaric acid (EDDG), or mixtures of any        of them, and    -   c. a pH of 5.8 or less;    -   d. wherein the beverage preservative system prevents spoilage by        microorganisms in a beverage within a sealed container for a        period of at least 16 weeks.

EDTA may need to be present where the purpose is to stabilize chemicalingredients.

The beverage preservative system also comprises a reverse sequestrant toa prescribed total amount wherein the composition contains any number oftypes of reverse sequestrant structures such that a total amount ofreverse sequestrant is achieved.

Example 14

According to another embodiment of the invention, a beveragepreservative system is provided which comprises:

-   -   a. sorbic acid, cinnamic acid, alkali metal salts thereof (e.g.,        K⁺, Na⁺), or mixtures of any of them that result in specific        concentrations of cinnamic or sorbic acid as determined by final        beverage pH;    -   b. a bio-degradable sequestrant selected from the group        consisting of ethylenediamine-N,N′-disuccinic acid (EDDS),        ethylenediamine-N,N′-dimalonic acid (EDDM),        ethylenediamine-N,N′-diglutaric acid (EDDG), or mixtures of any        of them, and    -   c. a pH of 5.8 or less;    -   d. wherein the beverage preservative system prevents spoilage by        microorganisms in a beverage within a sealed container for a        period of at least 16 weeks.

The beverage preservative system also comprises a reverse sequestrant toa prescribed total amount wherein the composition contains any number oftypes of reverse sequestrant structures such that a total amount ofreverse sequestrant is achieved.

The beverage preservative system also comprises an N-bis-phosphonate toa prescribed total amount wherein the composition contains any number oftypes of N-bis-phosphonate structures such that a total amount ofbis-phosphonate is achieved.

Example 15

According to another embodiment of the invention, a beveragepreservative system is provided which comprises:

-   -   a. sorbic acid, cinnamic acid, alkali metal salts thereof (e.g.,        K⁺, Na⁺), or mixtures of any of them that result in specific        concentrations of cinnamic or sorbic acid as determined by final        beverage pH;    -   b. a bio-degradable sequestrant selected from the group        consisting of ethylenediamine-N,N′-disuccinic acid (EDDS),        ethylenediamine-N,N′-dimalonic acid (EDDM),        ethylenediamine-N,N′-diglutaric acid (EDDG), or mixtures of any        of them, and    -   c. a pH of 5.8 or less;    -   d. wherein the beverage preservative system prevents spoilage by        microorganisms in a beverage within a sealed container for a        period of at least 16 weeks.

EDTA may need to be present where the purpose is to stabilize chemicalingredients.

The beverage preservative system also comprises a reverse sequestrant toa prescribed total amount wherein the composition contains any number oftypes of reverse sequestrant structures such that a total amount ofreverse sequestrant is achieved.

The beverage preservative system also comprises a bis-phosphonate to aprescribed total amount wherein the composition contains any number oftypes of bis-phosphonate structures such that a total amount ofbis-phosphonate is achieved.

Example 16

According to another embodiment of the invention, a beveragepreservative system is provided which comprises:

-   -   a. sorbic acid, cinnamic acid, alkali metal salts thereof (e.g.,        K⁺, Na⁺), or mixtures of any of them that result in specific        concentrations of cinnamic or sorbic acid as determined by final        beverage pH;    -   b. a bio-degradable sequestrant selected from the group        consisting of ethylenediamine-N,N′-disuccinic acid (EDDS),        ethylenediamine-N,N′-dimalonic acid (EDDM),        ethylenediamine-N,N′-diglutaric acid (EDDG), or mixtures of any        of them, and    -   c. a pH of 5.8 or less;    -   d. wherein the beverage preservative system prevents spoilage by        microorganisms in a beverage within a sealed container for a        period of at least 16 weeks.

EDTA may need to be present where the purpose is to stabilize chemicalingredients.

The beverage preservative system also comprises a reverse sequestrant toa prescribed total amount wherein the composition contains any number oftypes of reverse sequestrant structures such that a total amount ofreverse sequestrant is achieved.

The beverage preservative system also comprises a phosphonate orphosphonic acid to a prescribed total amount wherein the compositioncontains any number of types of phosphonate structures such that a totalamount of phosphonate is achieved.

Example 17

According to another embodiment of the invention, a beveragepreservative system is provided which comprises:

-   -   a. sorbic acid, cinnamic acid, alkali metal salts thereof (e.g.,        K⁺, Na⁺), or mixtures of any of them that result in specific        concentrations of cinnamic or sorbic acid as determined by final        beverage pH;    -   b. a bio-degradable sequestrant selected from the group        consisting of ethylenediamine-N,N′-disuccinic acid (EDDS),        ethylenediamine-N,N′-dimalonic acid (EDDM),        ethylenediamine-N,N′-diglutaric acid (EDDG), or mixtures of any        of them, and    -   c. a pH of 5.8 or less;    -   d. wherein the beverage preservative system prevents spoilage by        microorganisms in a beverage within a sealed container for a        period of at least 16 weeks.

EDTA may need to be present where the purpose is to stabilize chemicalingredients.

The beverage preservative system also comprises a reverse sequestrant toa prescribed total amount wherein the composition contains any number oftypes of reverse sequestrant structures such that a total amount ofreverse sequestrant is achieved.

The beverage preservative system also comprises Sodium Hexametaphosphate(SHMP), Sodium Acid Metaphosphate (SAMP), or mixture of SHMP and SAMP toa prescribed total amount.

Example 18

According to another embodiment of the invention, a beveragepreservative system is provided which comprises:

-   -   a. sorbic acid, cinnamic acid, alkali metal salts thereof (e.g.,        K⁺, Na⁺), or mixtures of any of them that result in specific        concentrations of cinnamic or sorbic acid as determined by final        beverage pH;    -   b. a bio-degradable sequestrant selected from the group        consisting of ethylenediamine-N,N′-disuccinic acid (EDDS),        ethylenediamine-N,N′-dimalonic acid (EDDM),        ethylenediamine-N,N′-diglutaric acid (EDDG), or mixtures of any        of them, and    -   c. a pH of 5.8 or less;    -   d. wherein the beverage preservative system prevents spoilage by        microorganisms in a beverage within a sealed container for a        period of at least 16 weeks.

EDTA may need to be present where the purpose is to stabilize chemicalingredients.

The beverage preservative system also comprises a reverse sequestrant toa prescribed total amount wherein the composition contains any number oftypes of reverse sequestrant structures such that a total amount ofreverse sequestrant is achieved.

The beverage preservative system also comprises an N-bis-phosphonate toa prescribed total amount wherein the composition contains any number oftypes of N-bis-phosphonate structures such that a total amount ofbis-phosphonate is achieved.

The beverage preservative system also comprises a bis-phosphonate to aprescribed total amount wherein the composition contains any number oftypes of bis-phosphonate structures such that a total amount ofbis-phosphonate is achieved.

Example 19

According to another embodiment of the invention, a beveragepreservative system is provided which comprises:

-   -   a. sorbic acid, cinnamic acid, alkali metal salts thereof (e.g.,        K⁺, Na⁺), or mixtures of any of them that result in specific        concentrations of cinnamic or sorbic acid as determined by final        beverage pH;    -   b. a bio-degradable sequestrant selected from the group        consisting of ethylenediamine-N,N′-disuccinic acid (EDDS),        ethylenediamine-N,N′-dimalonic acid (EDDM),        ethylenediamine-N,N′-diglutaric acid (EDDG), or mixtures of any        of them, and    -   c. a pH of 5.8 or less;    -   d. wherein the beverage preservative system prevents spoilage by        microorganisms in a beverage within a sealed container for a        period of at least 16 weeks.

EDTA may need to be present where the purpose is to stabilize chemicalingredients.

The beverage preservative system also comprises a reverse sequestrant toa prescribed total amount wherein the composition contains any number oftypes of reverse sequestrant structures such that a total amount ofreverse sequestrant is achieved.

The beverage preservative system also comprises an N-bis-phosphonate toa prescribed total amount wherein the composition contains any number oftypes of N-bis-phosphonate structures such that a total amount ofbis-phosphonate is achieved.

The beverage preservative system also comprises a phosphonate orphosphonic acid to a prescribed total amount wherein the compositioncontains any number of types of phosphonate structures such that a totalamount of phosphonate is achieved.

Example 20

According to another embodiment of the invention, a beveragepreservative system is provided which comprises:

-   -   a. sorbic acid, cinnamic acid, alkali metal salts thereof (e.g.,        K⁺, Na⁺), or mixtures of any of them that result in specific        concentrations of cinnamic or sorbic acid as determined by final        beverage pH;    -   b. a bio-degradable sequestrant selected from the group        consisting of ethylenediamine-N,N′-disuccinic acid (EDDS),        ethylenediamine-N,N′-dimalonic acid (EDDM),        ethylenediamine-N,N′-diglutaric acid (EDDG), or mixtures of any        of them, and    -   c. a pH of 5.8 or less;    -   d. wherein the beverage preservative system prevents spoilage by        microorganisms in a beverage within a sealed container for a        period of at least 16 weeks.

EDTA may need to be present where the purpose is to stabilize chemicalingredients.

The beverage preservative system also comprises a reverse sequestrant toa prescribed total amount wherein the composition contains any number oftypes of reverse sequestrant structures such that a total amount ofreverse sequestrant is achieved.

The beverage preservative system also comprises an N-bis-phosphonate toa prescribed total amount wherein the composition contains any number oftypes of N-bis-phosphonate structures such that a total amount ofbis-phosphonate is achieved.

The beverage preservative system also comprises Sodium Hexametaphosphate(SHMP), Sodium Acid Metaphosphate (SAMP), or mixture of SHMP and SAMP toa prescribed total amount.

Example 21

According to another embodiment of the invention, a beveragepreservative system is provided which comprises:

-   -   a. sorbic acid, cinnamic acid, alkali metal salts thereof (e.g.,        K⁺, Na⁺), or mixtures of any of them that result in specific        concentrations of cinnamic or sorbic acid as determined by final        beverage pH;    -   b. a bio-degradable sequestrant selected from the group        consisting of ethylenediamine-N,N′-disuccinic acid (EDDS),        ethylenediamine-N,N′-dimalonic acid (EDDM),        ethylenediamine-N,N′-diglutaric acid (EDDG), or mixtures of any        of them, and    -   c. a pH of 5.8 or less;    -   d. wherein the beverage preservative system prevents spoilage by        microorganisms in a beverage within a sealed container for a        period of at least 16 weeks.

EDTA may need to be present where the purpose is to stabilize chemicalingredients.

The beverage preservative system also comprises a reverse sequestrant toa prescribed total amount wherein the composition contains any number oftypes of reverse sequestrant structures such that a total amount ofreverse sequestrant is achieved.

The beverage preservative system also comprises a bis-phosphonate to aprescribed total amount wherein the composition contains any number oftypes of bis-phosphonate structures such that a total amount ofbis-phosphonate is achieved.

The beverage preservative system also comprises Sodium Hexametaphosphate(SHMP), Sodium Acid Metaphosphate (SAMP), or mixture of SHMP and SAMP toa prescribed total amount.

Example 22

According to another embodiment of the invention, a beveragepreservative system is provided which comprises:

-   -   a. sorbic acid, cinnamic acid, alkali metal salts thereof (e.g.,        K⁺, Na⁺), or mixtures of any of them that result in specific        concentrations of cinnamic or sorbic acid as determined by final        beverage pH;    -   b. a bio-degradable sequestrant selected from the group        consisting of ethylenediamine-N,N′-disuccinic acid (EDDS),        ethylenediamine-N,N′-dimalonic acid (EDDM),        ethylenediamine-N,N′-diglutaric acid (EDDG), or mixtures of any        of them, and    -   c. a pH of 5.8 or less;    -   d. wherein the beverage preservative system prevents spoilage by        microorganisms in a beverage within a sealed container for a        period of at least 16 weeks.

The beverage preservative system also comprises a reverse sequestrant toa prescribed total amount wherein the composition contains any number oftypes of reverse sequestrant structures such that a total amount ofreverse sequestrant is achieved.

The beverage preservative system also comprises an N-bis-phosphonate toa prescribed total amount wherein the composition contains any number oftypes of N-bis-phosphonate structures such that a total amount ofbis-phosphonate is achieved.

The beverage preservative system also comprises a bis-phosphonate to aprescribed total amount wherein the composition contains any number oftypes of bis-phosphonate structures such that a total amount ofbis-phosphonate is achieved.

The beverage preservative system also comprises a phosphonate orphosphonic acid to a prescribed total amount wherein the compositioncontains any number of types of phosphonate structures such that a totalamount of phosphonate is achieved.

Example 23

According to another embodiment of the invention, a beveragepreservative system is provided which comprises:

-   -   a. sorbic acid, cinnamic acid, alkali metal salts thereof (e.g.,        K⁺, Na⁺), or mixtures of any of them that result in specific        concentrations of cinnamic or sorbic acid as determined by final        beverage pH;    -   b. a bio-degradable sequestrant selected from the group        consisting of ethylenediamine-N,N′-disuccinic acid (EDDS),        ethylenediamine-N,N′-dimalonic acid (EDDM),        ethylenediamine-N,N′-diglutaric acid (EDDG), or mixtures of any        of them, and    -   c. a pH of 5.8 or less;    -   d. wherein the beverage preservative system prevents spoilage by        microorganisms in a beverage within a sealed container for a        period of at least 16 weeks.

The beverage preservative system also comprises a reverse sequestrant toa prescribed total amount wherein the composition contains any number oftypes of reverse sequestrant structures such that a total amount ofreverse sequestrant is achieved.

The beverage preservative system also comprises an N-bis-phosphonate toa prescribed total amount wherein the composition contains any number oftypes of N-bis-phosphonate structures such that a total amount ofbis-phosphonate is achieved.

The beverage preservative system also comprises a bis-phosphonate to aprescribed total amount wherein the composition contains any number oftypes of bis-phosphonate structures such that a total amount ofbis-phosphonate is achieved.

The beverage preservative system also comprises a phosphonate orphosphonic acid to a prescribed total amount wherein the compositioncontains any number of types of phosphonate structures such that a totalamount of phosphonate is achieved.

The beverage preservative system also comprises Sodium Hexametaphosphate(SHMP), Sodium Acid Metaphosphate (SAMP), or mixture of SHMP and SAMP toa prescribed total amount.

Example 24

Assuming compliance to upper limit constraints for the concentration ofCopper, Cobalt, Iron, Zinc, Aluminum, Chromium, and Nickel, the amountof preservatives disclosed herein that are required to stabilize abeverage are found to be dictated by the combined effect of two factors;the concentration of Calcium and the concentration of AssimilableNitrogen. Assimilable Nitrogen is principally an estimate of the reducedform of nitrogen (NH and NH₂) available in solution and serves as aestimate for the availability of those forms of nitrogen (ammonia, urea,amino acids and peptides) that can be employed as nutrients by thespoilage microorganism. A measure of total nitrogen would include allforms of nitrogen, some (Nitrate and Nitrite) which are not assimilableby fungi and many types of spoilage bacteria. The Table below disclosesexemplary concentration ranges for the various components in theexemplary beverage preservative systems described herein.

Formulation 0.26 mM < [Ca++] < 0.43 mM < [Ca++] < Requirements [Ca++] <0.26 mM 0.43 mM 0.764M TOTAL RANGE AN ≦ 3 ppm 10 ppm ≦ [WA] ≦ 50 ppm 10ppm < [WA] ≦ 50 ppm 10 ppm < [WA] ≦ 50 ppm 10 ppm < [WA] ≦ 50 ppm 0 ≦[EDTA] ≦ 45 0 ≦ [EDTA] ≦ 45 0 ≦ [EDTA] ≦ 45 0 [EDTA] ≦ 45 10 ppm < [BDS]≦ 30 ppm 10 ppm < [BDS] ≦ 3 0 ppm 10 ppm < [BDS] ≦ 45 ppm 10 pm < [BDS]≦ 45 ppm 0 ≦ [Phos] ≦ 150 0 ≦ [Phos] ≦ 600 0 ≦ [Phos] ≦ 1200 0 ≦ [Phos]≦ 1200 0 ≦ [Bis phos] ≦ 125 0 ≦ [Bis phos] ≦ 500 0 ≦ [Bis phos] ≦ 750 0≦ [Bis phos] ≦ 750 ≦ [N- bis phos] ≦ 125 0 ≦ [N- bis phos] ≦ 500 0 ≦ [N-bis phos] ≦ 750 0 ≦ [N- bis phos] ≦ 750 0 ≦ [R] ≦ 60 0 ≦ [R] ≦ 100 0 ≦[R] ≦ 150 0 < [R] ≦ 150 0 ≦ [SHMP] ≦ 600 0 ≦ [SHMP] ≦ 900 0 ≦ [SHMP] ≦1800 0 ≦ [SHMP] ≦ 1800 3 ppm < AN ≦ 10 ppm < [WA] < 650 ppm 10 ppm <[WA] < 650 ppm 10 ppm < [WA] < 650 ppm 10 ppm ≦ [WA] ≦ 650 ppm 10 ppm 0≦ [EDTA] < 45 0 ≦ [EDTA] < 45 0 [EDTA] < 45 0 ≦ [EDTA] ≦ 45 10 ppm <[BDS] < 45 ppm 10 ppm < [BDS] < 45 ppm 10 ppm < [BDS] < 45 ppm 10 ppm <[BDS] ≦ 45 ppm 0 ≦ [Phos] < 650 0 ≦ [Phos] < 950 0 < [Phos] < 1800 0 ≦[Phos] ≦ 1250 0 ≦ [Bis phos] < 750 0 ≦ [Bis phos] < 750 0 < [Bis phos] <750 0 ≦ [Bis phos] ≦ 1500 [N- bis phos] < 750 0 ≦ [N- bis phos] < 750[N- bis phos] < 750 0 ≦ [N- bis phos] ≦ 1500 0 ≦ [R] < 60 0 ≦ [R] < 60 0< [R] < 60 0 ≦ [R] ≦ 60 0 ≦ [SHMP] < 1800 0 ≦ [SHMP] < 1800 0 ≦ [SHMP] <1800 0 ≦ [SHMP] ≦ 1800 10 ppm < AN ≦ 10 ppm < [WA] ≦ 850 ppm 10 ppm <[WA] ≦ 850 ppm 10 ppm ≦ [WA] ≦ 850 ppm 10 ppm ≦ [WA] ≦ 850 ppm 50 ppm 0[EDTA] ≦ 75 0 [EDTA] ≦ 75 0 ≦ [EDTA] ≦ 75 0 ≦ [EDTA] ≦ 75 10 ppm < [BDS]≦ 75 ppm 10 ppm < [BDS] ≦ 75 ppm 10 ppm ≦ [BDS] ≦ 75 ppm 10 ppm ≦ [BDS]≦ 75 ppm 0 ≦ [Phos] ≦ 1200 0 ≦ [Phos] ≦ 1500 0 ≦ [Phos] ≦ 2100 0 ≦[Phos] ≦ 1800 0 ≦ [Bis phos] ≦ 800 0 ≦ [Bis phos] < 1200 0 ≦ [Bis phos]< 1800 0 ≦ [Bis phos] < 1800 ≦ [N- bis phos] ≦ 800 ≦ [N- bis phos] ≦1200 ≦ [N- bis phos] ≦ 1800 800 ≦ [N- bis phos] ≦ 1800 0 ≦ [R] ≦ 75 0 ≦[R] ≦ 75 0 ≦ [R] ≦ 100 0 ≦ [R] ≦ 100 0 ≦ [SHMP] < 1800 0 ≦ [SHMP] < 18000 ≦ [SHMP] < 1800 0 ≦ [SHMP] < 1800 AN = Assimilable Nitrogen WA = weakacid EDTA = ethylene diamine tetraacetic aci d BDS = bio degradablesequestrant Phos = phosphonate Bis-phos = Bis phosphonate N-bis Phos =N-bis phosphonate R = Reverse sequestrant SHMP = Sodiumhexametaphosphate OR Sodium acid metaphosphate (SAMP) -- interchangeable

In the following examples, the added water had/has a hardness less than50 ppm and alkalinity less than 50 ppm. Copper, less than 1 microgramper liter Zinc less than 1 microgram per liter, Nickel, less than 1microgram per liter and Iron less than 0.1 microgram per liter. Thewater hardness was/is adjusted to 50 ppm through addition of mixture ofCaCl₂ and MgCl₂.

The spoilage organisms for the following examples are identified asfollows: Y3, Zygosaccharomyces bailii isolate of Pepsi (Strain 906);C-7UP, Brettanomyces isolate of Pepsi, (Cherry 7-UP); Spore, ascosporepreparation of Saccharomyces cerevisiae isolate of Pepsi (strain“spore”). Y22, ATCC 60484 Zygosacharomyces bailii; M1, Paecilomycesisolate of Pepsi; Y107 ATCC 52407; M2 ATCC 36614 Byssochlamys nieva. Aninoculum of approximately 40 per milliliter was/is employed for eachorganism. All test samples were/are incubated 16 weeks at 25 C.

Example 25

The following example provides an illustration of the hurdles associatedwith the preservation of beverages through the use of weak organicacids. A 2 fold concentrate of beverage was prepared and was then mixedwith an equal volume of 15% molten agar. Before solidification of theagar, the single strength beverage formulation containing 7.5% moltenagar, was then divided into aliquots and to each separate aliquot wasadded, with the exception of one, sodium benzoate, potassium sorbate orcinnamic acid. The aliquot not receiving either potassium sorbate orcinnamic acid served as a positive control. While the agar was stillmolten, the agar-beverage aliquots (containing a single weak acid at aspecified concentration) were poured into separate Petri plates. In Twoagar plates. sodium benzoate was present at either 700 ppm or 1000 ppm)Two agar plates contained potassium sorbate (500 or 800 ppm). Six agarplates contained cinnamic acid ranging in concentrations of 25-200 ppm.Agar solidification essentially provides single strength beverageformulation in a semi-solid state.

The 2 fold concentrate of single strength beverage and the agarsupplemented single strength beverage is prepared as follows:

Sucrose 64 g Glucose 52 g Fructose 2 g Apple Juice: 20 ml SingleStrength Malic acid 0.67 g Na + Malate 0.268 g (Sodium Malate) WaterBring volume to 500 ml 15% molten agar Mix 1:1 with beverage formulationPotassium Sorbate 0.07 or 0.10% (700 & 1000 ppm) if Benzoate or Cinnamicacid not present Sodium Benzoate 0.05 and 0.08% (500 & 800 ppm) ifPotassium sorbate or Cinnamic acid not present Cinnamic acid0.0025-0.02% (25, 35, 50, 100, 150, and 200 ppm) if Potassium Sorbateand Sodium Benzoate not present

After solidification of juice-agar mixture, various yeast strains (FIG.2A) were spotted to the gel-surface of each type of mixture employing anidentical pattern in each test (FIG. 2B) such that it was possible toidentify the outgrowth of specific individual strains. The controlplate, which did not contain any organic acid preservative (FIG. 2C),showed that most strains grew reasonably well. FIG. 2C also demonstratesthat the different yeast strains are not all equally tolerant to organicacid preservatives. Strain Y3 (formal id strain 906 in-house collection)proved to be among strains demonstrating tolerance to both benzoic acidand sorbic acid. Y3 is a Zygossacharomyces bailii strain (DNA homology)isolated from spoiled beverage. Strain Y3 also proved to be among themost tolerant strains to Cinnamic acid (FIG. 2D).

Example 26

A preparation of beverage was divided into multiple small volumealiquots, each aliquot containing a different concentration of potassiumsorbate. The range of concentration of potassium sorbate is 0-0.094%(0-940 ppm). Preparations (aliquots) of beverage containing potassiumsorbate at each test concentration (48 different values) are inoculatedseparately with one of 7 different spoilage organisms (bio-indicator)for a total of 336 test samples.

The 2% fruit juice based non-carbonated beverage of pH 3.4 and about 12Brix was generated by combining the following ingredients.

Added Water Approximately 84% water Apple Juice About 0.372% to providesingle Concentrate strength concentration of about 2% Sucrose 6.8%Glucose 5.2% Fructose 0.2% Potassium Sorbate 0-0.094% Malic acid0.0674%   Sodium Malate 0.013% (approximately, adjusting pH to 3.4)Ethylene diamine 30 ppm in test sample (adjusted for tetraacetic acid(EDTA) M++ present in salt) Ethylene diamine 15 or 30 ppm in test sample(adjusted disuccinic acid (EDDS) for M++ present in salt)

The test design established the minimum inhibitory concentration ofpotassium sorbate to preserve a product of pH 3.4 against differentclasses of spoilage organisms in the absence of a physical agent such asheat or irradiation. Additionally, the small increment of potassiumsorbate concentration in the test series allowed some estimate of thevariability of tolerance to potassium sorbate among the bio-indicatorstrains. A sequestrant such as EDTA or EDDS can complement potassiumsorbate additively or synergistically by either inhibiting a cellfunction separate of that from sorbate or by working in concert withpotassium sorbate through some function that reduces variability oftolerance within a population. The results of the test are found in FIG.3A. FIG. 3B serves to facilitate interpretation of FIG. 3A.

FIG. 3A demonstrates that the range of tolerance to potassium sorbate isquite large across the group of bio-indicators but that the majority ofthe variability in the group is driven by strain Y3, a Zygosaccharomycesbailii strain. The data, serves to emphasize the generally recognizedfact that some strains of Zygosaccharomyces bailli are measurablytolerant to weak acid preservatives such as potassium sorbate. StrainY3, an isolate of spoiled commercial beverage, was the most tolerantstrains of weak acid preservative from among nearly 100 yeast strainstested. In the absence of Y3, the amount of potassium sorbate requiredto preserve product might be as low as 220 to 250 ppm. Although 220-250ppm potassium sorbate is a preferable concentration over the 550-600concentration required to inhibit Y3 or similar strains, it ismeasurably high with regard to its implications to the sensory profileof a beverage. Further, at least one regulatory agency in Asia hasestablished an upper limit for the use of potassium sorbate at 50 ppm.If other regulatory agencies follow suite, it will be necessary tobroadly apply a complement to potassium sorbate. Finally, potassiumsorbate is not particularly soluble at acid pH and it has a measurablylow rate of dissolution. Beverages are generally produced fromconcentrates and a 4 fold beverage concentrate should contain 1200-1500ppm potassium sorbate to yield a finished beverage concentration of240-300 ppm. This often proves problematic during the batching ofbeverage and it is clear that a final beverage formulation containing50-150 ppm potassium sorbate would be favored.

FIG. 3A also possesses data that draws attention to the variance amongindividual members within the population of organisms introduced intothe test samples (inoculum). Between the ranges of potassium sorbateconcentrations of 460-660 ppm some test samples into which strain Y3 wasinoculated were free of growth only for the beverage containingconsecutively greater concentrations of sorbate to be supportive ofgrowth. This observation is most easily explained by the likelihood thatthe standard deviation for tolerance to sorbic acid is measurablygreater for this strain of Z. bailii than is commonly observed for otherspoilage organisms, such as those strains other than Y3 that are used asbio-indicators in this study.

It should be noted that the although ppm is often an appropriate term toquantify the amount of substance added to food or beverage that thebiochemistry of preservation is driven by the number of preservativemolecules present which is most correctly expressed in terms ofmolarity. The difference between 500 ppm potassium sorbate and 800 ppmin terms of molarity is less than 2 mM (5.3 mM-3.3 mM). A standarddeviation of 75 pm equates to 0.49 mM (or 0.00049M). As suggested byFIG. 3B, the expectation is to find instances where growth occurs in asample that contains an incrementally higher concentration ofpreservative to a sample in which no growth occurred. The analysis ofFIG. 3B is not a statistical summary of data from FIG. 3A and merelyserves to identify the most likely explanation for the observed patternof growth and no growth demonstrated in FIG. 3A and other such studies.

Example 27

The following example provided an illustration of the preservativecapacity of beverage product of the present invention when EDDS iscombined with a single weak acid preservative (potassium sorbate). Apreparation of beverage was divided into three aliquots; one wassupplemented with EDTA (30 ppm) and the remaining two aliquots weresupplemented with either 15 or 30 ppm EDDS.

A 2% fruit juice based non-carbonated beverage of pH 3.4 and about12Brix was formed by combining the following ingredients.

Added Water Approximately 84% water Apple Juice About .3.72% to providesingle Concentrate strength concentration of about 2% Sucrose 6.8%Glucose 5.2% Fructose 0.2% Potassium Sorbate 0-0.15% Malic acid0.0674%   Sodium Malate 0.013% (approximately, adjusting pH to 3.4)Ethylene diamine 30 ppm in test sample (adjusted for M++ tetraaceticacid (EDTA) present in salt) if EDDS not present Ethylene diamine 15 or30 ppm in test sample (adjusted for M++ disuccinic acid (EDDS) presentin salt) if EDTA is not present

The beverage aliquots were identical with the exception of the presenceor concentration of either EDTA or EDDS. Each aliquot was dividedfurther into smaller volume test samples wherein the concentration ofpotassium sorbate varies from 0-1500 ppm (0-0.15%). The concentration ofEDTA or EDDS is held constant. Under these circumstances, it is possibleto establish whether either EDTA or EDDS permitted the use of loweredconcentrations of potassium sorbate to preserve a product of pH 3.4. Ifthe minimum inhibitory concentration of potassium sorbate differedsignificantly in one preparation over the other, then it was presumedthat EDTA or EDDS provided different complement to potassium sorbatewith regard to inhibition of outgrowth of spoilage organisms. If the twosubstances proved equivalent complement to potassium sorbate, EDDS wasthe favored substance for use in beverage formulation because it is amore environmentally sound application. The beverage is formulated tocontain 50 ppm hardness. Consequently, the amount of calcium andmagnesium measurably exceed the binding capacity of either EDTA or EDDSfor these molecules. However, either EDTA or EDDS were able to bindother cations such as copper and zinc.

Data revealed in FIGS. 4A-4C suggest that EDDS is equivalent to EDTA asa preservative when combined with a single weak acid preservative. FIG.4A is data for the combination of EDTA and potassium sorbate wherein theconcentration of EDTA was constant at 30 ppm and the concentration ofpotassium sorbate is varied from 0-940 ppm. FIG. 4B is data for thecombination of EDDS (30 ppm) and potassium sorbate. As in FIG. 4A, theconcentration of EDDS was held constant while the concentration ofpotassium sorbate was varied from 0-920 ppm. The data set for FIG. 4Crepresents the interaction between 15 ppm EDDS and potassium sorbate.The data in the three sets of experiments was collected in parallel withexample 24. Comparing the two examples (3a & 4a-c) reveals that thepresence of EDDS or EDTA complemented potassium sorbate with regard tomold, but offered some relief to the concentration of potassium sorbaterequired for inhibition of growth by yeast fungi. Mold (M1 & M6) provedto be inhibited by a combination of potassium sorbate (140-160 ppm) andeither EDTA or EDDS (30 ppm) wherein the required concentration ofpotassium sorbate was lower than if potassium sorbate was employed alone(180-200 ppm).

The small, but clear advantage offered by the addition of EDDS or EDTAis fully expected given the chemistry of EDTA and EDDS. Further, it wasexpected that both substances would perform similarly. Both substancessequester Fe+++, Cu++, Zn++, Mn++ in exactly the same order ofpreference. Although EDDS exhibited slightly lower binding constants, itwas still measurably in excess of what is required given theconcentration of such trace elements typically present in beverage.Although EDDS and EDTA were chemically equivalent in regard to theirfunction as sequestrants, EDDS was preferred because it is less of anaffront to the environment. In fact, there is published evidence thatEDDS is itself a natural substance.

Example 28

The following example provides an illustration of the preservativecapacity of beverage product of the present invention. The biodegradablesequestrant ethylene diamine disuccinic acid was admixed with a sodiumhexametaphosphate and cinnamic acid in the beverage as shown in theformula for the beverage.

A 2% fruit juice based non-carbonated beverage of pH 3.4 and about 12Brix was formed by combining the following ingredients.

Added Water Approximately 84% water Apple Juice About 0.372% to providesingle Concentrate strength concentration of about 2% Sucrose 6.8%Glucose 5.2% Fructose 0.2% Potassium Cinnamate 0 to 0.06% (o to 6oo ppm)Malic acid 0.067%  Sodium Malate 0.013% (approximately, adjusting pH to3.4) Ethylene diamine 30 ppm, if present in test sample tetraacetic acid(EDTA) Ethylene diamine 30 ppm, if present in test sample disuccinicacid (EDDS) Sodium 0.075% (750 ppm) hexametaphosphate (SHMP) Hardness 50ppm total

FIG. 5A data demonstrates that 30 ppm EDTA can combine with 750 ppm SHMPand as little as 75 ppm K+cinnamate and to prohibit outgrowth of all 7bio-indicator strains. An identical beverage formulation in which EDDSis substituted for EDTA (FIG. 5B) yielded similar result wherein 30 ppmEDDS combined with 750 ppm SHMP and no more than 50 ppm K+cinnamate toprohibit the outgrowth of all 7 bio-indicator strains.

Combining EDDS and SHMP apparently serves to limit the availability ofone or more divalent cations critical to the outgrowth to spoilagemicroorganisms and weakens them toward the effect of weak acids. Forexample, it is known that ethylene diamine disuccinic acid served tobind iron, copper and other trace elements whereas sodiumhexametaphosphate binds Calcium and Magnesium. The preservative systemis, apparently, dependent on the combined activity of ethylene diaminedisuccinic acid, Sodium Hexametaphosphate and a weak acid.

Example 29

The following example provides an illustration of the preservativecapacity of beverage product of the present invention. The biodegradablesequestrant ethylene diamine disuccinic acid was admixed with abis-phosphonate (Etidronate) wherein the two sequestrants combined tolimit the availability of divalent cations to spoilage microorganisms.The beverage was also supplemented with potassium sorbate. The combinedaction of sequestrants served to reduce the concentration of potassiumsorbate required to prohibit outgrowth of spoilage organisms. There islittle difference in the capacity of mixtures of EDDS and Etidronaterelative to traditional mixtures of Ethylene diamine tetra acetic acid(EDTA) and sodium hexametaphosphate (SHMP) to complement Potassiumsorbate.

A 2% fruit juice based non-carbonated beverage of pH 3.4 and about12Brix was formed by combining the following ingredients.

Added Water Approximately 84% water Apple Juice About 0.372% to providesingle Concentrate strength concentration of about 2% Sucrose 6.8%Glucose 5.2% Fructose 0.2% Potassium Sorbate 0.015%  Malic acid0.0674%   Sodium Malate 0.013% (approximately, adjusting pH to 3.4)Ethylene diamine 30 ppm in test sample, if EDDS not present tetraaceticacid (EDTA) Ethylene diamine 30 ppm in test sample, if EDTA not presentdisuccinic acid (EDDS) Sodium 0-1500 ppm hexametaphosphate (SHMP)Bis-phosphonate, 0-1500 ppm Etidronate

Data in FIG. 6A demonstrates that 30 ppm EDDS combined with as little as280 ppm SHMP to prohibit outgrowth of all 7 bio-indicator strains. Anidentical beverage formulation in which EDTA was substituted for EDDS(FIG. 6B) yielded a comparable result wherein 30 ppm EDTA combined with250 ppm SHMP to prohibit the outgrowth of all 7 bio-indicator strains.

The combination of 30 ppm EDDS and <425 ppm of the bis-phosphonate,Etridonate complemented a concentration of 150 ppm potassium sorbate toprohibit the outgrowth of all seven bio-indicator strains (FIG. 6C). Thecombination of 30 ppm EDDS and 250 ppm Etridonate served to complement150 ppm potassium sorbate in the prevention of outgrowth of all sevenspoilage organisms (FIG. 6D).

The combination of 30 ppm EDDS and <450 ppm of the bis-phosphonate,Etridonate can complement a concentration of 150 ppm potassium sorbateto prohibit the outgrowth of all seven bio-indicator strains. The choiceof slightly elevated concentrations of Etridonate in the presence ofEDDS offered the option of employing the bio-degradable sequestrantEDDS.

Furthermore, the problem of pH drift that accompanies the use of SHMP isavoided if Etidronate is substituted for SHMP. Substitution of EDDS forEDTA avoids the concerns associated with employing a substance that isnot environmentally sound

Example 30

The following example provides an illustration of the projectedpreservative capacity of beverage product of the present invention. Thebiodegradable sequestrant ethylene diamine disuccinic acid (50 ppm) isadmixed with 150 ppm potassium sorbate and 30 ppm Cinnamic acid into a2% juice beverage that is carbonated (2.5-3.5 volumes of CO₂). Incommercial operations, carbonation of a product occurs immediately priorto its packaging into either a glass, plastic or aluminum container orother such vessel that is generally impervious to oxygen ingress. In theabsence of EDDS, the beverage formulated containing 500 ppm potassiumsorbate will yield to spoilage before the end of targeted shelf life (16weeks). The combination of 50 ppm EDDS and 500 ppm potassium sorbatewill permit the 2% juice beverage initially containing 2.5-3.5 volumesof CO₂ to remain stable against spoilage for a period of at least 16weeks.

A 2% fruit juice based non-carbonated beverage of pH 3.4 and about 12Brix is formed by combining the following ingredients.

Added Water Approximately 84% water Apple Juice About 0.372% to providesingle Concentrate strength concentration of about 2% Sucrose 6.8%Glucose 5.2% Fructose 0.2% Potassium Sorbate 0.015%  Cinnamic acid0.003%  Malic acid 0.0674%   Sodium Malate 0.013% (approximately,adjusting pH to 3.4) EDDS 50 ppm Carbon Dioxide 2.5 to 3.5 volumes (1volume = 1.96 g CO₂ per liter)

Example 31

Beverages processed by standard “hot-fill” are typically filled into aheat-set PET container that can accommodate product temperatures ofabout 85° C. The heat contained in the product filled in the containerserves to kill omnipresent fungal spores that either associated withbeverage contact surfaces of the package material or settled intoproduct in the period between filling and closure application.Commercial and environmental considerations favor the development of aformulation or process that would allow the use of light weight plasticcontainers to fill thermally processed beverage by standard “hot-fill”process design. However containers made from light weightnon-crystalline PET rapidly deforms if the temperature of the plasticexceeds 71.3° C., the glass transition temperature of PET.

As evidenced by data summarized in FIGS. 7A, 7B, and 7C, the temperatureof product immediately before filling into a standard PET containercannot exceed 76.6° C. (170° F.) if the bottle wall temperature is notto exceed 71.1° C. (160° F.). Under such conditions, the highesttemperature to which organisms are exposed is 73-74° C. (168° F.) and itis possible that many organisms adhering to wall of container mayexperience temperatures no greater than 150-160° F. (65.5-71.1 C). It isalso apparent that the temperature profile drops quickly in the 2minutes before product in container enters the cooling tunnel.

FIG. 7B indicates that many common mold spores can withstand exposure of71° C. for a period of at least 4 to 6 minutes. As indicated in FIG. 6A,the amount of time that product is actually at 71° C. is less than 15-30seconds. It is standard practice in hot-fill operations to begin coolingof product 2 minutes after cooling in preparation for the loading ofpackage into case packs. It is not practical or economically feasible toalter the time period between fill and cooling operations. In FIG. 7Cdata is shown which demonstrates that many types of common mold sporesurvive a process that mimics a hot-fill of 71° C. product. Herein,heated product was filled into container into which mold spores had beeninoculated. After 2 minutes, the test vessels were immersed into ambientwater to mimic passage through cooling tunnel. Nearly all the types ofmold spores tested germinated and evolved into mycelium (+).

The dilemma of the restriction of heat content in product relative tosurvival of mold spores is circumvented by employing the invention in anapplication that can be termed “hybrid”. Hybrid is the combination ofphysical and chemical agents to preserve product. In this instance, heatcontained in the beverage is employed to rid the package material ofvegetative forms of yeast, bacteria and the invention is employed toprohibit the outgrowth of mold spores that are present in the containerat the time of closure application and that are able to survive theexposure to product filled into the container at temperature of 71°C.±3° C. The temperature of product in this hybrid “hot-fill” allows theuse of standard light weight PET. Further, the invention is heat stableand there is no loss of activity of the invention when exposed topasteurization and fill temperatures. Polyphosphates, if present, willhydrolyze causing changes to pH and sensory attributes of the product.This fact is not widely understood among experts in the field ofbeverage formulation or microbiology. Less appreciated still is the heatstability of the di-phosphonates.

The example for hybrid process that incorporates the invention employsthe beverage formulation shown below. The biodegradable sequestrantEthylene Diamine Disuccinic acid (30 ppm) is admixed into 2% juicebeverage with 500 ppm of Etridonate. The 2% juice product is also madeto contain a combination of weak organic acid (cinnamic acid andpotassium sorbate at a combined concentration of 100 to 150 ppm. Theproduct is then heat pasteurized and then filled into a container suchthat neither the in package temperature of product or body of thecontainer exceeds 71.3° C. It has been demonstrated that combinationheat contained in product filled into container at 71.3° C. incombination with just weak acids totaling 100 ppm is insufficient toprohibit the outgrowth of mold spores from many type of fungi. (FIG.7C).

The maximum temperature of product in container of 71.3° C. permits theuse of standard PET containers as opposed to the environmentallyless-sound heat-set PET containers typically employed in hot filloperations. The combination of weak acid preservative, 30 ppm EDDS (orEDTA) and 500-1500 ppm bis-phosphonate (Etridonate) serve to prevent theoutgrowth of spores of spoilage fungi that either were associated withthe product contact surface of the package vessel prior to filling orthat settled into the beverage in the short time frame between thefilling and sealing of containers. A formulation containing SHMP inplace of bis-phosphonate will fail because of the degradation of SHMPthat occurs during thermal processing.

To date, there are no regulatory agency stated limits for the use ofcinnamic acid, but cinnamic acid has a taste threshold of about 30-35ppm and so the lower the concentration of cinnamic acid, the better theproduct will be received by the consumer. One or more countries haverecently set restrictions on the upper limit of potassium sorbate to nomore than 50 ppm.

A 2% fruit juice based non-carbonated beverage of pH 3.4 and about12Brix is formed by combining the following ingredients.

Added Water Approximately 84% water Apple Juice About 0.372% to providesingle Concentrate strength concentration of about 2% Sucrose 6.8%Glucose 5.2% Fructose 0.2% Potassium Sorbate 0.015%  Malic acid0.0674%   Sodium Malate 0.013% (approximately, adjusting pH to 3.4)Ethylene diamine 30 ppm in test sample, unless EDDS present tetraaceticacid (EDTA) Ethylene diamine 30 ppm in test sample, unless EDTA presentdisuccinic acid (EDDS) Sodium 500-1500 ppm, unless Etidronate presenthexametaphosphate (SHMP) Bis-phosphonate, 500-1500 ppm, unless SHMP ispresent Etidronate

Etidronate, as a substitute for SHMP, allows for the use of thermalprocessing of beverage in that Etidronate will not acid hydrolyze as isthe case for SHMP. Furthermore, the problem of pH drift that accompaniesthe use of SHMP is avoided. Substituting EDDS for EDTA avoids theconcerns associated with employing a substance that is notenvironmentally sound.

Example 32

The invention is particularly suited to beverage formulations that arewithin the category of nutraceuticals. Such products include energydrinks, flavored water and tea beverages. Healthy and good-for-youformulations of beverages often require that ingredients are eithernatural or provide function as nutraceuticals. It is already understoodthat bis-phosphonates, such as Etidronate, can function to preventtartar build-up on teeth (a nutraceutical function). It was largelyunexpected that these same compounds proved effective as complements toweak acid preservatives in the prevention growth of spoilage bacteria orfungi. The finding that EDDS serves to complement the activity ofbis-phosphonates when combined with weak acid preservatives is alsounexpected and fortuitous given the fact that EDDS is bothbio-degradable and a naturally formed substance. Note that naturalsources of sorbic acid and cinnamic acid exist and that the syntheticforms employed in test are natural identical.

Prior to initial tests with bis-phosphonates, it was not immediatelyapparent that these substances were capable of sequestering sufficientquantities of divalent cations to a degree that would impact theobserved tolerance (lowering) to weak acid preservatives. The initialstudies with Etidronate proved that bis-phosphonates had the potentialfor use in some if not most beverage formulations. The similarity ofstructure among the bis-phosphonates dictates very similar chemistry forthese substances in regard to their ability to bind di-valent cations(M++) and to do so across a range of pH. Consequently, most, if not all,bis-phosphonate can readily substitute for another bis-phosphonate in abeverage formulation and the choice of bis-phosphonate would be basedlargely on issues such as solubility, sensory threshold or cost ofingredient. The beverage formulation that is the basis of this exampleis employed to demonstrate the interchangeable nature of thebis-phosphonates wherein the substitution of one bis-phosphonates foranother does not serve to alter the observed stability of product givena set amount of either cinnamic acid or potassium sorbate (examples ofweak acid and salts of weak acids).

Water 94.44 g Sugars, total 4.5 g Minerals Calcium, Ca 17 mg Iron, Fe <3μg Magnesium, 3.0 mg Sodium, Na 8   Zinc, Zn 0.32 Copper, Cu 0.007 mgVitamins Vitamin C, total 12.7 mg ascorbic acid Niacin 0.844 mg VitaminB-6 0.084 mg Folate, total mcg 8 0 8 μg Vitamin B-12 0.25 μg Vitamin AInternational unit of 106 Retinol mcg 32 0 32 μg Vitamin E (alpha- 1.9mg tocopherol) mg 1.90 0 Vitamin B-12, added Vitamin A, RAE mcg_RAE 32 0mcg 0.25 0 Preservative Substances Sorbic acid 50-200 mg Cinnamic acid50-200 mg Etidronate 500 ppm or 0 ppm if other bis- phosphonate ispresent Ibandronic acid 500 ppm or 0 ppm if other bis- phosphonate ispresent Medronic acid 500 ppm or 0 ppm if other bis- phosphonate ispresent Clodronic acid 500 ppm or 0 ppm if other bis- phosphonate ispresent Alendronic acid 500 ppm or 0 ppm if other bis- phosphonate ispresent Pamidronic acid 500 ppm or 0 ppm if other bis- phosphonate ispresent EDDS 45 ppm

Bis-phosphonates, as a substitute for SHMP, allows for the use ofthermal processing of beverage in that all bis-phosphonates are stableagainst heat or acid induced hydrolysis. Such is not the case forpolyphosphates such as SHMP. Furthermore, the problem of pH drift thataccompanies the use of SHMP at ambient temperatures is avoided.Substituting EDDS for EDTA avoids the concerns associated with employinga substance that is not environmentally sound.

All example formulations used to establish efficacy of the preservativesystem are identical except for variation about the type ofdi-phosphonate that is present. Positive control samples are identicalin formulation except for the presence preservatives. Positive controlsinclude formulation with no weak acid preservative, a formulation withjust sorbic acid (varying concentrations) with just cinnamic acid(varying concentrations) a formulation with 45 ppm EDDS and varyingconcentrations of sorbic acid and formulation with 45 ppm EDDS andvarying concentrations of Cinnamic acid and a formulation with no weakacid preservative and only 45 ppm EDDS.

The presence of each of the bis-phosphonates will reduce theconcentration of weak acid preservative that is required to prohibitspoilage by any of the following bio-indicator organisms.

Example 33

A N′ bis-phosphonate is the sequestrant of choice for a beverage to beconsumed by adults and distributed and sold in equatorial regions of theworld. Phosphonates are temperature stable and their use is favored overpolyphosphates in instances where product is either subject to thermalprocessing or is exposed to rather harsh temperatures (excess of 25° C.)for excessive periods (days or weeks) during distribution and display.For example, N′ bis-phosphonate is the preferred sequestrant fornon-alcoholic malt beverages that are extremely popular among adultpopulations in equatorial regions of the world where refrigerateddistribution and display are lacking. In such regions, it is not out ofthe ordinary for product to be subjected temperatures in the range of33-35° C. (91-95° F.) for extended periods of time. In the instance ofthis example, it can be demonstrated that Malt beverage product exposedto temperatures of 33° C. for even a period of 4 weeks is more stablewhen formulated with N′ bis-phosphonate than polyphosphate. N′bis-phosphonate offer the added benefit of functioning as anutraceutical or a “good for you” ingredient in this particularformulation that is most likely to be consumed by adults. N-bisphosphonates are particularly adept at preventing bone re-adsorption,the cause of osteoporosis.

Finally, an unexpected discovery is that N′ bis-phosphonates will retardgrowth of spoilage organisms in and of themselves when added to abeverage formulation wherein the pH is between 2.8 and 5.5. When presentat a concentration of about 300 ppm in a beverage acidified to pH 3.6with malic acid, Ibandronate will impair the growth of Saccharomycescerevisae. When present at 0.3% concentration in the same beverageformulation, Alendronate retards the growth rate of Saccharomycescerevisae. It is speculated, not to be bound by theory, that N-bisphosphonates are themselves antifungal in the range of acid pH and, to adegree and may function by interfering with certain metabolic pathwaysfound in fungi or eukaryotic organisms in general.

Concentrations of N bis-phosphonate below 100 ppm will complement theactivity of traditional weak acid preservatives to completely inhibitthe outgrowth of spoilage organisms. The combination of N-bisphosphonateand weak acid preservative can be balanced for taste such that theconcentration of N-bis phosphonate does not need to exceed 100 ppm andthat lowest concentration of weak acid or weak acid salt is present. Thefinding that EDDS further serves to complement the activity of differentN-bis-phosphonates in combination with weak acids allows the formulationof beverages that will remain stable against spoilage frommicroorganisms for a period of at least 16 weeks, even when held attemperature extremes of 35° C. for the entire shelf life. In many partsof Middle East and Africa where refrigerated display is lacking, a shelfstable product must be able to tolerate such temperatures for extendedperiods.

The beverage formulation that is the basis of this example can beemployed to demonstrate the interchangeable nature of the Nbis-phosphonates wherein the substitution of N-bis-phosphonates foranother does not serve to alter the observed stability of product givena set amount of either cinnamic acid or potassium sorbate (examples ofweak acid and salts of weak acids). The beverage is generally, speaking,targeted to adults in the population and so there is a possible “healthand wellness” attribute to because of the presence ofN′bis-phosphonates. The benefit from consuming such a product must beestablished (absorbed dose from gut).

Water, 91.15 g Malt (est by FAN) 0.21 g Fiber, total dietary g 0.02 gSugars, total 8.05 g Minerals Calcium, Ca 7 mg Iron, Fe 0.06 mgMagnesium, Mg 7 mg Phosphorus, P 16 mg Potassium, K 8 mg Sodium, Na 13mg Zinc, Zn mg 0.02 mg Copper, Cu 0.008 mg Manganese, 0.013 mg Selenium,1.2 μg Vitamins Vitamin C, total ascorbic acid 0.5 mg Thiamin 0.016 mgRiboflavin 0.048 mg Niacin mg 1.113 mg Pantothenic acid 0.037 mg VitaminB-6 0.027 mg Folate 14 μg Choline, total mg 10.1 0 10 μg Vitamin B-12mcg 0.02 0 0.02 μg Lipids Fatty acids, total saturated g 0.024 g 16:00.021 g 18:0 0.001 g Fatty acids, total 0.056 g polyunsaturated Alcohol,ethyl 0.3 g Icadronate 0-500 ppm, 0 if other N′bis- phosphonate ispresent Alendronate 0-500 ppm, 0 if other N′bis- phosphonate is presentPamidronate 0-500 ppm, 0 if other N′bis- phosphonate is presentNeirdronate 0-500 ppm, 0 if other N′bis- phosphonate is present EDTA 30ppm Potassium Sorbate 125 ppm

N′ Bis-phosphonates, as a substitute for SHMP, allows for the use ofthermal processing of beverage in that all N′bis-phosphonates are stableagainst heat or acid induced hydrolysis. Such is not the case forpolyphosphates such as SHMP. Furthermore, the problem of pH drift thataccompanies the use of SHMP at ambient temperatures is avoided.Substituting EDDS for EDTA avoids the concerns associated with employinga substance that is not environmentally sound.

All example formulations used to establish efficacy of the preservativesystem are identical except for variation about the type of N′bis-phosphonate that is present. Positive control samples are identicalin formulation except for the presence preservatives. Positive controlsinclude formulation with no weak acid preservative, a formulation withjust sorbic acid (varying concentrations) with just cinnamic acid(varying concentrations) a formulation with 45 ppm EDDS and varyingconcentrations of sorbic acid and formulation with 45 ppm EDDS andvarying concentrations of cinnamic acid and a formulation with no weakacid preservative and only 45 ppm EDDS.

The presence of each of the N′bis-phosphonates will reduce theconcentration of weak acid preservative that is required to prohibitspoilage.

Example 34

A bis-phosphonate is employed as a sequestrant for a tea beverage.Although N′ bis-phosphonates are generally stronger sequestrants thanare bis-phosphonates, there are formulations wherein bis-phosphonatesprove a more appropriate sequestrant. For instance, it was unexpectedlydiscovered that some N′ bis-phosphonates, but not bis-phosphonates,result in the formation of a precipitate and haze when added to teabeverage. Although not wanting to be bound by theory, it appears thatthe positive charge associated with N′ bis-phosphonates is responsiblefor this phenomenon. Given the weaker sequestrant activity ofbis-phosphonates relative to N′ bis-phosphonates the ability ofbis-phosphonate to complement weak acid preservatives in tea was afavorable and not fully expected result.

Although bis-phosphonates, such as Etidronate, do not function toprevent bone re-adsorption to the same degree as N′bis-phosphonates,they do function to prevent tartar build-up on teeth. Consequently, theuse of bis-phosphonates still offers the option of a nutraceuticalfunction in instances where this single function is most appropriate.

The example shown here builds on the earlier cited examples whereinmicrobiological evidence is provided that Etidronate, a bis-phosphonate,serves to function as a complement to potassium sorbate in thepreservation of a juice based product formulation. As is the case withN′ bis-phosphonate, a similarity of structure among the differentbis-phosphonates allows a certain amount of interchangeability amongbis-phosphonates allowing product developers a choice of sequestrantbased on sensory or cost attributes. The beverage formulation that isthe basis of this example is employed to demonstrate the interchangeablenature of the bis-phosphonates wherein the substitution of onebis-phosphonates for another does not serve to alter the observedstability of product given a set amount of either cinnamic acid orpotassium sorbate (examples of weak acid and salts of weak acids).

The finding that EDDS serves to complement the activity ofbis-phosphonates when combined with weak acid preservatives in an acidicmedium is measurably unexpected and fortuitous given the fact that EDDSis both bio-degradable and a naturally formed substance. Its similarityin structure to a peptide may have resulted in some measure of acidhydrolysis. Note that natural sources of Sorbic acid and Cinnamic acidexist and that the synthetic forms employed in test are naturalidentical.

Water 91.06 g Tea solid 0.22 g Carbohydrate 8.81 g Fructose 0.5 g Pectin0.165 g Rebaudioside A .2 g Lemon Flavor 2.0 g Citric acid .5 g Na +Citrate 0.1 g Calcium, Ca 1 mg Iron, Fe .001 Magnesium, Mg 0 mgPhosphorus, P 26 mg Potassium, K 19 mg Sodium, Na 21 mg Zinc, Zn .01 mgCopper, Cu .005 mg Manganese, Mn 0.146 mg Fluoride, F 72.2 mcg Caffeine2 mg Sorbic acid 50-200 mg Cinnamic acid 50-200 mg Etidronate 500 ppm or0 ppm if other bis-phosphonate is present Ibandronic acid 500 ppm or 0ppm if other bis-phosphonate is present Medronic acid 500 ppm or 0 ppmif other bis-phosphonate is present Clodronic acid 500 ppm or 0 ppm ifother bis-phosphonate is present Alendronic acid 500 ppm or 0 ppm ifother bis-phosphonate is present Pamidronic acid 500 ppm or 0 ppm ifother bis-phosphonate is present EDDS 45 ppm

Bis-phosphonates, in place of SHMP, allows for the use of thermalprocessing of beverage in that all bis-phosphonates are stable againstheat or acid induced hydrolysis. Such is not the case for polyphosphatessuch as SHMP. Furthermore, the problem of pH drift that accompanies theuse of SHMP at ambient temperatures is avoided. Substituting EDDS forEDTA avoids the concerns associated with employing a substance that isnot environmentally sound.

All example formulations to establish efficacy of the preservativesystem are identical except for variation about the type ofdi-phosphonate that is present. Positive control samples are identicalin formulation except for the presence preservatives. Positive controlsinclude formulation with no weak acid preservative, a formulation withjust sorbic acid (varying concentrations) with just cinnamic acid(varying concentrations) a formulation with 45 ppm EDDS and varyingconcentrations of sorbic acid and formulation with 45 ppm EDDS andvarying concentrations of cinnamic acid and a formulation with no weakacid preservative and only 45 ppm EDDS.

The presence of each of the N′ bis-phosphonates will reduce theconcentration of weak acid preservative that is required to prohibitspoilage.

Example 35

Phosphonic acid is used as a sequestrant preservative for sportsbeverages and other formulations that make “good for you” claims. Theinvention is particularly in keeping with the concept of a beveragenutraceutical wherein the claim is a replenishment of electrolytes lostduring exertion.

The formulation of energy drinks, flavored water and tea beverages isoften an exercise in optimization wherein the beverage must deliveragainst label claims, possess an acceptable taste, and also maintainstability against microbial induced spoilage for over the period of theproduct shelf life. As a rule of thumb, sequestrants typically do notbind measurable amounts of mono-valent cations such as sodium andpotassium, but the stronger the sequestrant the more likely is aninteraction will occur. For instance, EDTA binds sodium in a manner thatthe ratio of bound to unbound is over 10:1 in mildly acid medium (log K25 C is 1.7). Polyphosphates can bind both sodium and potassium inmeasurable amounts at higher pH values as evidenced by availability ofcommercial products that are composed polyphosphate bound to both Sodiumand Potassium in various ratios.

The phosphonates and amino-phosphonates are not nearly as potentsequestrants as are di-phosphonates or bis-phosphonates orpolyphosphate. As such, the phosphonates can be combined with lowconcentrations of EDTA or EDDS in a manner that avoids measurablybinding of either Sodium or Potassium. This fact is so broadlyappreciated by those practiced in the art that a claim about electrolyteavailability would not necessitate proof by way of human or animalstudies. In combination with EDDS (principally binding trace metal ionsrequired by organism for growth such as Iron and copper), thephosphonates function quite well in combination with weak acidpreservatives, such as Cinnamic acid to preserve a sports beverage. Bothphosphonate and amino-phosphonate are stable at elevated temperaturesand can be employed in hybrid hot fill processes as described in example30.

As is the case for the bis-phosphonates, different Phosphonates (oramino Phosphonates) can be used interchangeably depending on ingredientcost, solubility issues or sensory preferences. Phosphonates, as asubstitute for SHMP, allows for the use of thermal processing ofbeverage in that all bis-phosphonates are stable against heat or acidinduced hydrolysis. Such is not the case for polyphosphates such asSHMP. Furthermore, the problem of pH drift that accompanies the use ofSHMP at ambient temperatures is avoided. Substituting EDDS for EDTAavoids the concerns associated with employing a substance that is notenvironmentally sound and allows for the possibility of naturalsubstance claim.

All example formulations used to establish efficacy of the preservativesystem are identical except for variation about the type of phosphonate(or amino-phosphonate) that is present. Positive control samples areidentical in formulation except for the presence preservatives. Positivecontrols include formulation with no weak acid preservative, aformulation with just sorbic acid (varying concentrations) with justCinnamic acid (varying concentrations) a formulation with 45 ppm EDDSand varying concentrations of sorbic acid and formulation with 45 ppmEDDS and varying concentrations of Cinnamic acid and a formulation withno weak acid preservative and only 45 ppm EDDS.

Water 91.91 Ash 0.2 Sucrose 0.2 g Glucose (dextrose) 2.20 g Fructose3.22 g Lactose 0.2 g Maltose 0.2 g Minerals Calcium, Ca 1   Iron, Fe0.09 mg Magnesium, Mg <0.02 mg Phosphorus, P 1 mg Potassium, K 18 mgSodium, Na 22 mg Zinc, Zn 0.01 mg Copper, Cu <0.01 mg Manganese, Mn<0.001 mg Fluoride, F 62 mg Vitamins Thiamin 0.011 mg Niacin 1.56 mgVitamin B-6 0.153 mg Vitamin B-12 1.37 mg PRESERVATIVE Cinnamic acid0-200 ppm EDDS 45 ppm 2 hydroxy 600 ppm or 0 ppm if otherphosphonoacetic acid phosphonate present (HPAA) phenylphosphonic acid600 ppm or 0 ppm if other (PPA) phosphonate present Aminotri methylene600 ppm or 0 ppm if other phosphonic acid phosphonate presentN-phosphonacetyl-:L- 600 ppm or 0 ppm if other aspartate (PALA)phosphonate present

The presence of each of the bis-phosphonates will reduce theconcentration of weak acid preservative that is required to prohibitspoilage by the bio-indicator organisms Y3, C-7Up, Spore, Y22, M1, Y107,and M2.

Example 35

A lipid soluble sequestrant (reverse sequestrant) is used in combinationwith a bis-phosphonate and weak acid organic acid to preserve either aflavor or cloud emulsion. It is the nature of emulsions to exist asspherical droplet like structures when in aqueous suspension wherein thecore of the droplet is hydrophobic and is able to solubilize lipidsoluble (aqueous insoluble) components such as flavors, colors,nutrients, and cloud components. Typically, emulsion droplets are on theorder of 1-3 micron diameter. The stability of emulsions againstspoilage requires the presence of preservative in a measurable amount inthe aqueous phase. However, weak organic acids tend to concentrate intothe non-aqueous phase of the emulsion. The difference in concentrationbetween aqueous phase and emulsion may be as much as lo fold asestablished from log P (or log D) estimates.

It has recently come to our attention that sorbic acid and cinnamic acidare prone to oxidative degradation and that the rate of degradation canbe slowed by the addition of appropriate amounts of sequestrants tomixtures containing the weak acids. EDTA, when present in the aqueousphase, suffices to protect against oxidation. However, in an emulsion,the weak acid preservatives concentrate into the non-aqueous phase andEDTA is not measurably soluble in the non-aqueous phase. The problem canbe addressed by employing lipid soluble (reverse sequestrants) as acomponent of the emulsion. Herein, a Ca++ specific reverse sequestrantsuch as BAPTA (1,2-Bis(2-Aminophenoxy)ethane-N,N,N,′N′-tetraacetic acid,or BAPTA-AM (1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acidtetrakis (acetoxymethyl ester) is deployed in conjunction with the Ironspecific reverse sequestrant known as Exochelin to stabilize an emulsionagainst spoilage. EDTA and either a bis-phosphonate or N′bis-phosphonateare also incorporated into the formula for the emulsion in order toallow some protection in the aqueous phase.

Not to be bound by theory, it appears that the reverse phasesequestrants serve to prohibit attack by spoilage organisms towardsmaller emulsion particles (possibly by a endocytotic mechanisms) andalso serve to protect weak acids from oxidation. The double bond ofsorbic acid is as prone to oxidation as are double bonds in fatty acids,a factor that leads to rancidity. Because the weak acids concentrateabout 10 fold into the non-aqueous phase, there is less of a requirementfor the sequestrants to complement weak acid preservative antimicrobialactivity in this portion of the emulsion. Complement to theantimicrobial activity is forthcoming via the Ca++ sequestrant. The ironbinding Exochelin actually serves to protect against oxidation of weakorganic acid preservatives.

The aqueous phase is protected by a lower concentration of weak acidpreservative in the presence of a sizeable amount of Ca+ sequesteringactivity via EDTA in combination with either a bis-phosphonate orN′bis-phosphonate. The concentration of components in the emulsionreflects the fact that emulsions are prepared as concentrates and thefinal concentration of emulsion components in beverage is rather low. Itis not the intent to preserve the finished beverage with just thepreservative components added to the emulsion.

A cloud emulsion containing bis-phosphonate and the reverse sequestrantsBAPTA-AM and Exochelin.

RO treated Water 84.9% Modified Starch (Octenyl Succinate) 7.5 g/LCottonseed oil 6.5 g/L Ascorbic acid 0.05 g/L Natural Color 0.05 g/LCitric acid 0.75 g/L Potassium Sorbate 0.25 g/L Reverse SequestrantBAPTAM-AM 2.3 g (50 mM) (1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethyl ester) Exochelin (natural frommycobacterium) 150 ppm EDTA 150 ppm 1-hydroxyethane-1,1-diphosphonicacid (HEDP) 750 ppm

An orange flavor emulsion containing bis-phosphonate and BAPTA-AMreserve sequestrant.

RO treated Water 85.8% Ethyl alcohol 0.313 g/L Acetylaldehyde 0.384 g/LAscorbic acid .575 g/L Sodium Citrate 0.74 g/L Sodium Benzoate 1.02/LCitric acid 1.57 g/L Glycerol Ester of Wood Rosin 32.3 g/L Flavor 36 g/LFood Starch 110 g//L Reverse Sequestrant PCIH (2- 2.3 g (50 mM)pyridylcarboxaldehyde isonicotinoyl hydrazone) Exochelin 150 ppm EDDS150 ppm Alendronate (N′bis-phosphonate) 750 ppm

Example 37

The following example is a projected embodiment of the invention anddemonstrates the use of reverse sequestrants, EDDS, and eitherN′bis-phosphonate or bis-phosphonate in the generation of a beveragethat contains a flavor, cloud or flavor/cloud emulsion.

U.S. Pat. No. 5,641,532 discloses that the presence of polyphosphates,especially sodium hexametaphosphate, can be destabilizing to aflavor/cloud emulsion used in various beverages. According to U.S. Pat.No. 5,641,532, the destabilizing effect likely reflects an interactionbetween polyphosphate and the emulsion particle that causes exclusion ofthe emulsion from the aqueous phase and results in enhanced aggregationand flocculation of the emulsion pa. U.S. Pat. No. 5,641,532 alsoteaches a solution to this problem, wherein the solution permits (1) thegeneration of a stable emulsion (2) the generation of beveragecontaining a stable emulsion containing polyphosphate (3) a beveragewith acceptable mouth feel and (4) a beverage with no undesirable flavoreffects. The solution for the problem is outlined by way of examples (1and 2).

Example 1 of U.S. Pat. No. 5,641,532 requires the heating of 1.69 g SHMPsuspension in water to a temperature of 37.8° C. (100° F.) to forcedissolution of SHMP. This SHMP solution is mixed with gellan gum toproduce a SHMP/gellan gum suspension referred to as a “SHMP/thickenersuspension”. The “SHMP/thickener suspension” is then blended into asolution of juice concentrate and flavor/cloud emulsion. In this manner,a finished beverage is finally derived containing approximately 1000 ppmSHMP. A second embodiment of the invention outlines the generation of aflavor or cloud emulsion, the preparation of a “thickener” composed of aspecified mixture of SHMP and carboxymethylcellulose, and the blendingof the SHMP/thickener and the base beverage into single strengthbeverage that requires pasteurization at 186.0° F. It is not indicatedwhether the flavor or cloud emulsion is added before or after thethermal processing step, but in that the HTST step is typically employedas a “terminal” process, it can be assumed that the emulsion is addedprior to HTST.

The following provides a process and formulation for the generation of abeverage containing a flavor or cloud emulsion and that is preserved bya combination of weak organic acids (or salts or weak organic acids) incombination with EDDS or EDTA and a bis-phosphonate or N′ bisphosphonate in accordance with at least one aspect of the invention.

A cloud emulsion is prepared containing bis-phosphonate and the reversesequestrants BAPTA-AM and Exochelin.

Ingredient Amt RO treated Water 84.9% Octenyl Succinate modified starch(emulsifier) 7.5 g/L Canola (clouding agent) 6.5 g/L Ascorbic acid 0.05g/L Natural Color 0.05 g/L Citric acid 0.75 g/L Potassium Sorbate 0.25g/L Reverse Sequestrant BAPTAM-AM 0.23 g (5 mM) (1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′- tetraacetic acid tetrakis (acetoxymethyl ester)Exochelin (natural from mycobacterium) 300 ppm EDTA 300 ppm1-hydroxyethane-1,1-diphosphonic acid (HEDP) 300 ppm

The above ingredients are then homogenized. The emulsion can be storedat ambient for a period of months or can be immediately batched with aconcentrate that has been prepared as follows. Note that the concentrateis itself quite stable against spoilage because it contains over 500 ppmPotassium Sorbate, >150 ppm sequestrant.

Ingredient % RO treated Water 56.64 Citric Acid 7.75 Sodium Citrate 1.4Vitamin B1 0.002 Juice Concentrate 13.0 Cloud Emulsion 21.0 Flavor 0.2

Assuming that the concentrate is blended into final beverage at or about8% (standard practice) It is important to note that the preservativescontained in the concentrate carried over to finish beverage atconcentrations that need not be declared on the label of the product.

Ingredient Quantity RO treated Water 78.91%  Vitamin C supplement 0.04%Sweetener  13% Beverage concentrate   8% K + sorbate 4.2 ppm HEDP 5.0ppm EDTA 5.0 ppm BAPTAM-AM 3.8 ppm Exochelin 5.0 ppm

The final beverage formulation after the blending of emulsion withbeverage concentrate can be tailored to contain various preservatives atconcentrations that are consistent with the threat from various classesof spoilage organisms. A beverage that is to be carbonated will not beinclined to spoil from mold fungi but is at risk of spoilage fromvarious yeast and bacteria such as Lactobacillus, Brettanomyces andSaccharomyces. A batched product that is to be thermally processed andfilled into container vessel at temperatures that will ensure thedestruction of vegetative yeast and bacteria might still be at some riskof spoilage from germinating mold spores if the fill temperature is lessthan 850 C (standard for hot-fill). Per way of example, the finalbeverage formulation is prepared. Prior to the adjustment ofpreservatives that would reflect the nature of the terminal step inprocessing, the concentration of substances in batched product are asfollows”

This example offers a solution toward the prevention of spoilage of theemulsion. Emulsions are prone to spoilage and are routinely made tocontain preservatives to allow for a prescribed shelf life. According toU.S. Pat. No. 5,641,532, it is not possible to enhance the activity ofweak organic acids in flavor/cloud emulsions with polyphosphates becauseof the propensity of polymeric structures such as polyphosphates tocompromise the dispersion of emulsions. N′bis-phosphonates andbis-phosphonates are not polymeric and the complexes formed betweenPhosphonates and cations are measurably non-polymeric in structure (2-3phosphonates serve to “encage” a divalent cation). The presence of N′bis-phosphonate or bis-phosphonates in the emulsion in combination withthe weak organic acids serves to extend the shelf-life of an emulsionbeyond what is possible employing no preservative (U.S. Pat. No.5,641,532) or employing weak acid preservatives alone. Often, flavor andcloud emulsions are prepared, stored and shipped from locations that arequite remote from the location of the blending and bottling of thefinished product. Given the costly ingredients present in flavor orcloud emulsions, it is imperative that emulsion stability be extended aslong as possible.

Another advantage of the present invention is that a “thickener” is nota required component of the final beverage formulation. U.S. Pat. No.5,641,532 teaches that an absolute requirement for the “thickener” abeverage formulation that also contains polyphosphate and either a cloudor flavor emulsion. Further, there is an order of addition forsubstances that requires at least 3 separate steps. U.S. Pat. No.5,641,532 teaches that the thickener (per way of example, Gellan gum,gelatin, or carboxymethylcellulose) serves to stabilize the emulsionagainst the tendency to coalesce when in the presence of polyphosphate.Although the reason for the ordered addition of ingredients is notprovided, it is likely that a three step process is dictated by theconcern that the emulsion can “shock out” if brought into contact withlocalized high concentrations of polyphosphates (as would occur whenblending ingredients).

The invention described herein provides the option of whether to add a“thickener” and also serves to simplify the batching operation. A flavoror cloud emulsion containing a preservative system composed of eitherEDDS or EDTA in combination with either a bis-phosphonate or N′bis-phosphonate is simply added to the beverage “base” wherein the baseis principally composed of sweetener, acidulant, other preservatives andwater. The product developer has the option to add a “thickener”. Thisis a more cost effective process, requiring less equipment, time andhuman resource.

The processes cited in examples of U.S. Pat. No. 5,641,532 requireexposure of polyphosphates to a thermal process. In example 1, the blendof thickener and polyphosphate is exposed to a temperature of 100° F.for an unspecified period of time. In example 2, the now dilute blend ofpolyphosphate/polyphosphate is thermally processed by HTST at atemperature of 190° F. for unspecified number of seconds before coolingto 60° F. over an unspecified period of time. In both instances, anamount of polyphosphate will be hydrolyzed and this will result inchanges to both pH and mouth-feel. The rather excessive concentration of1000-3000 ppm SHMP employed in the beverage formulations likely reflectthe loss of polyphosphate concentration during processing.

In accordance with the present invention, because N′ bis-phosphonate andbis-phosphonates are stable to thermal processing these substances neednot be “over dosed” into product in order to ensure their presence attargeted concentrations.

Given the benefit of the above disclosure and description of exemplaryembodiments, it will be apparent to those skilled in the art thatnumerous alternative and different embodiments are possible in keepingwith the general principles of the invention disclosed here. Thoseskilled in this art will recognize that all such various modificationsand alternative embodiments are within the true scope and spirit of theinvention. The appended claims are intended to cover all suchmodifications and alternative embodiments. It should be understood thatthe use of a singular indefinite or definite article (e.g., “a,” “an,”“the,” etc.) in this disclosure and in the following claims follows thetraditional approach in patents of meaning “at least one” unless in aparticular instance it is clear from context that the term is intendedin that particular instance to mean specifically one and only one.Likewise, the term “comprising” is open ended, not excluding additionalitems, features, components, etc.

Example 38

The invention is particularly suited to beverage formulations that arewithin the category of nutraceuticals and for which an all natural claimis to be made. Such products include energy drinks, flavored water andtea beverages. Healthy and good-for-you formulations of beverages oftenrequire that ingredients are either natural or provide function asnutraceuticals. From a previous example, it is already understood thatPhosphonates and N-phosphonates, such as 2-aminoethylphosphonic acid orN-(phosphonoacetyl)-L-aspartame can be employed as complements to weakorganic acids and EDDS or EDTA in order to preserve certain classes ofproducts.

Further, it is understood that N-phosphonates and Phosphonates serve toinhibit tartar build-up on teeth (a nutraceutical function). It waslargely unexpected that these same compounds proved effective ascomplements to weak acid preservatives in the prevention growth ofspoilage bacteria or fungi. The finding that EDDS serves to complementthe activity of phosphonates and N-phosphonates when combined with weakacid preservatives is also unexpected and fortuitous given the fact thatEDDS is both bio-degradable and a naturally formed substance. Note thatnatural sources of sorbic acid and cinnamic acid exist and that thesynthetic forms employed in test are natural identical.

Further, natural Phosphonates and amino-phosphonates exist and thesesubstances function similar to the synthetic forms of these compounds.

Thus, it is possible to formulate a chemically preserved beverage inwhich all components of the preservative system are naturally derived orare naturally identical substances

The similarity of structure among the phosphonates and amongamino-phosphonates dictates very similar chemistry for these substancesin regard to their ability to bind di-valent cations (M++) and to do soacross a range of pH. Consequently, most, if not all, Phosphonates andamino-phosphonates can readily substitute for another in a beverageformulation and the choice of phosphonate or amino-phosphonate would bebased largely on issues such as availability as a naturally derivedsubstance, sensory threshold or cost of ingredient. The beverageformulation that is the basis of this example is employed to demonstratethe interchangeable nature of the bis-phosphonates wherein thesubstitution of one bis-phosphonates for another does not serve to alterthe observed stability of product given a set amount of either cinnamicacid or potassium sorbate (examples of weak acid and salts of weakacids).

Water 94.44 g Sugars, total 4.5 g Minerals Calcium, Ca 17 mg Iron, Fe <3μg Magnesium, 3.0 mg Sodium, Na 8   Zinc, Zn 0.32 Copper, Cu 0.007 mgVitamins Vitamin C, total ascorbic acid 12.7 mg Niacin 0.844 mg VitaminB-6 0.084 mg Folate, total mcg 8 0 8 μg Vitamin B-12 0.25 μg Vitamin AInternational unit of 106 Retinol mcg 32 0 32 μg Vitamin E(alpha-tocopherol) 1.9 mg mg 1.90 0 Vitamin B-12, added mcg 0.25 0Vitamin A, RAE mcg_RAE 32 0 Preservative Substances Sorbic acid 50-200ppm, unless other weak acid or salt present Cinnamic acid 50-200 ppm,unless other weak acid or salt present K + sorbate 75-250 ppm, unlessother weak acid or salt present K + Cinnamate 75-250 ppm unless otherweak acid or salt present Sorbic acid, Cinnamic acid, Combinedconcentration not K + cinnamic acid or K + to exceed 250 ppm sorbate2-aminoethylphosphonic acid Singularly or in combination with other(AEP) N-phosphosphonate totaling ≦750 ppm Fosfomycin Singularly or incombination with other N-phosphosphonate totaling ≦750 ppmPhosphinothricin Singularly or in combination with otherN-phosphosphonate totaling ≦750 ppm 2-methylaminoehtylophosphonicSingularly or in combination with other acid (N,N dimethyl AEP)N-phosphosphonate totaling ≦750 ppm 2- Singularly or in combination withother trimethylaminoethylphosphonic N-phosphosphonate totaling ≦750 ppmacid 2 amino-3-phosphonopropionic Singularly or in combination withother acid N-phosphosphonate totaling ≦750 ppm EDDS 45 ppm

Phosphonates or N-phosphonates, as a substitute for SHMP, allows for theuse of thermal processing of beverage in that all phosphonates arestable against heat or acid induced hydrolysis. Such is not the case forpolyphosphates such as SHMP. Furthermore, the problem of pH drift thataccompanies the use of SHMP at ambient temperatures is avoided.Substituting EDDS for EDTA avoids the concerns associated with employinga substance that is not environmentally sound.

All example formulations used to establish efficacy of the preservativesystem are identical except for variation about the type ofN-phosphonate that is present. Positive control samples are identical informulation except for the presence preservatives. Positive controlsinclude formulation with no weak acid preservative, a formulation withjust sorbic acid (varying concentrations) with just cinnamic acid(varying concentrations) a formulation with 45 ppm EDDS and varyingconcentrations of sorbic acid and formulation with 45 ppm EDDS andvarying concentrations of Cinnamic acid and a formulation with no weakacid preservative and only 45 ppm EDDS.

The presence of each of the N-phosphonates will reduce the concentrationof weak acid preservative that is required to prohibit spoilage by anyof the following bio-indicator organisms.

1-19. (canceled)
 20. A beverage comprising: a beverage component; abeverage preservative system comprising: an N-bis-phosphonic acid oralkali metal salt thereof; a biodegradable sequestrant at aconcentration in a range of 10 ppm to 120 ppm, wherein the biodegradablesequestrant is selected from the group consisting ofethylenediamine-N,N′-disuccinic acid (EDDS),ethylenediamine-N,N′-dimalonic acid (EDDM),ethylenediamine-N,N′-diglutaric acid (EDDG) and mixtures of any of; anda weak acid selected from the group consisting of cinnamic acid, sorbicacid, their alkali metal salts, and mixtures of any of them; wherein thebeverage has a pH of 5.8 or less; and wherein the beverage when placedwithin a sealed container is substantially not spoiled by microorganismsfor a period of at least 16 weeks.
 21. The beverage of claim 20, whereinthe beverage component is selected from the group consisting of addedwater, a juice, a flavorant, a sweetener, an acidulant, a colorant, avitamin, a buffering agent, a thickener, an emulsifier, an anti-foamingagent, and mixtures of any of them.
 22. The beverage of claim 20,wherein the beverage is selected from the group consisting of acarbonated beverage, a non-carbonated beverage, a soft drink, a fruitjuice, a fruit juice flavored drink, a fruit-flavored drink, an energydrink, a hydration drink, a sport drink, a health and wellness drink, afountain beverage, a frozen ready-to-drink beverage, a frozen carbonatedbeverage, a liquid concentrate, a coffee beverage, a tea beverage, adairy beverage, a soy beverage, a vegetable drink, a flavored water, anenhanced water, and an alcoholic beverage.
 23. The beverage of claim 20,wherein the beverage preservative system further comprises a reversesequestrant selected from the group consisting of:1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA),1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acidtetraacetoxymethyl ester (MAPTAM),N,N,N′,N′-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN), exochelin,pyridoxal isonicotinoyl hydrazone (PIH), 2-pyridylcarboxaldehydeisonicotinoyl hydrazone (PCIH), di-2-pyridylketone isonicotinoylhydrazone (PKIH), 2-quinolinecarboxaldehyde isonicotinoyl hydrazone(QCIH), 2-pyridylcarboxaldehyde 2-thiophenecarboxyl hydrazone (PCTH),di-2-pyridylketone 2-thiophenecarboxyl hydrazone (PKTH),2-quinolinecarboxaldehyde 2-thiophenecarboxyl hydrazone (QCTH),2-pyridylcarboxaldehyde m-bromobenzoyl hydrazone (PCBBH),2-pyridylcarboxaldehyde benzoyl hydrazone (PCBH), di-2-pyridylketonebenzoyl hydrazone (PKBH), 2-quinolinecarboxaldehyde benzoyl hydrazone(QCBH), 2-pyridylcarboxaldehyde p-aminobenzoyl hydrazone (PCAH),di-2-pyridylketone p-aminobenzoyl hydrazone (PKAH),2-quinolinecarboxaldehyde p-aminobenzoyl hydrazone (QCAH),2-pyridylcarboxaldehyde p-hydroxylbenzoyl hydrazone (PCHH),di-2-pyridylketone p-hydroxylbenzoyl hydrazone (PKHH),2-quinolinecarboxaldehyde p-hydroxylbenzoyl hydrazone (QCHH),2-furoylcarboxaldehyde isonicotinoyl hydrazone (FIH), and mixtures ofany of them.
 24. The beverage of claim 20, wherein the N-bis-phosphonicacid is selected from the group consisting of: pamidronate, neridronate,olpadronate, risedronate, alendronate, zoledronate, ibandronate,incadronate, minodronate, piperidin-1-yl-methane-1,1-diphosphonic acidand derivatives thereof having at least one methyl or ethyl substituenton the piperidinyl ring, and mixtures of any of them.
 25. The beverageof claim 20, wherein the beverage preservative system further comprisesa polyphosphate selected from the group consisting of sodiumhexametaphosphate (SHMP), sodium acid metaphosphate (SAMP), and mixturesthereof.
 26. The beverage of claim 20, wherein the beverage has a pH of4.4 or less.
 27. The beverage of claim 20, wherein the beverage has a pHin a range of 2.6 to 3.8.
 28. The beverage of claim 20, wherein thebeverage preservative system comprises the biodegradable sequestrant ata concentration of 10 ppm to 75 ppm.
 29. The beverage of claim 23,wherein the beverage preservative system comprises the reversesequestrant at a concentration of 150 ppm or less.
 30. The beverage ofclaim 20, wherein the beverage preservative system further comprisesEDTA at a concentration of 75 ppm or less.
 31. The beverage of claim 20,wherein the beverage preservative system comprises the N-bis-phosphonicacid or alkali metal salt thereof at two to three times a combined molarconcentration of magnesium and calcium cations present in the beveragepreservative system.
 32. The beverage of claim 20, wherein the beveragepreservative system comprises the N-bis-phosphonic acid or alkali metalsalt thereof at a concentration of 1800 ppm or less.
 33. The beverage ofclaim 20, wherein the beverage preservative system comprises the weakacid at a concentration in the range of 10 ppm to 850 ppm.
 34. Thebeverage of claim 20, wherein the beverage preservative system furthercomprises added water that has been treated by reverse osmosis,electro-deionization or both to decrease a total concentration of metalcations of chromium, aluminum, nickel, zinc, copper, manganese, cobalt,calcium, magnesium, and iron to 1.0 mM or less.
 35. The beverage ofclaim 20, wherein metal cations of chromium, aluminum, nickel, zinc,copper, manganese, cobalt, calcium, magnesium, and iron are present inthe beverage preservative system at a total concentration of 1.0 mM orless.